Methods for nucleic acid sequencing

ABSTRACT

Methods of sequencing molecules based on luminescence lifetimes and/or intensities are provided. In some aspects, methods of sequencing nucleic acids involve determining the luminescence lifetimes, and optionally luminescence intensities, of a series of luminescently labeled nucleotides incorporated during a nucleic acid sequencing reaction.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 62/164,482, titled “METHODS FORNUCLEIC ACID SEQUENCING,” filed May 20, 2015, U.S. Provisional PatentApplication No. 62/164,506, titled “INTEGRATED DEVICE FOR TEMPORALBINNING OF RECEIVED PHOTONS,” filed May 20, 2015, U.S. ProvisionalPatent Application No. 62/164,464, titled “INTEGRATED DEVICE WITHEXTERNAL LIGHT SOURCE FOR PROBING, DETECTING, AND ANALYZING MOLECULES,”filed May 20, 2015, and U.S. Provisional Patent Application No.62/164,485, titled “PULSED LASER,” filed May 20, 2015, each of which ishereby incorporated by reference in its entirety.

This application claims priority under 35 U.S.C. §120 to U.S. patentapplication Ser. No. 14/821,688, titled “INTEGRATED DEVICE WITH EXTERNALLIGHT SOURCE FOR PROBING, DETECTING, AND ANALYZING MOLECULES,” filedAug. 7, 2015, which is hereby incorporated by reference in its entirety.

FIELD OF THE APPLICATION

The present application is directed generally to methods, compositions,and devices for performing rapid, massively parallel, quantitativeanalysis of biological and/or chemical samples, and methods offabricating said devices.

BACKGROUND

Detection and analysis of biological samples may be performed usingbiological assays (“bioassays”). Bioassays conventionally involve large,expensive laboratory equipment requiring research scientists trained tooperate the equipment and perform the bioassays. Moreover, bioassays areconventionally performed in bulk such that a large amount of aparticular type of sample is necessary for detection and quantitation.

Some bioassays are performed by tagging samples with luminescent markersthat emit light of a particular wavelength. The markers are illuminatedwith a light source to cause luminescence, and the luminescent light isdetected with a photodetector to quantify the amount of luminescentlight emitted by the markers. Bioassays using luminescent markersconventionally involve expensive laser light sources to illuminatesamples and complicated luminescent detection optics and electronics tocollect the luminescence from the illuminated samples.

SUMMARY

According to an aspect of the present application, a single molecule canbe identified (e.g., distinguished from other possible molecules in areaction sample) based on one or more properties of a series of photonsthat are emitted from the molecule when it is exposed to a plurality ofseparate light pulses. In some embodiments, the emitted photons can beused to identify/distinguish the molecule based on a differentluminescent lifetime. In some embodiments, luminescent lifetime can becalculated. In some embodiments, the actual lifetime does not need to becalculated. For example, one or more properties suggestive ofluminescent lifetime can be used (e.g., local time of emission, timedistribution of detected emissions). In some embodiments, the emittedphotons can be used to determine the luminescent intensity of themolecule, and the luminescent intensity can be used to identify themolecule. In some embodiments, the emitted photons can be used todetermine the luminescent lifetime and luminescent intensity of themolecule, and the luminescent lifetime or luminescent intensity can beused to identify the molecule. In some embodiments, the identity of themolecule can be based on a combination of both luminescent lifetime andluminescent intensity.

In some embodiments, a molecule can be labeled with a luminescent label.In some embodiments, the luminescent label is a fluorophore. In someembodiments, the luminescent label can be identified or distinguishedbased on a property of the luminescent label. Properties of aluminescent label (e.g., a fluorophore) include, but are not limited toluminescent lifetimes, absorption spectra, emission spectra,luminescence quantum yield, and luminescent intensity. In someembodiments, luminescent labels are identified or distinguished based onluminescent lifetime. In some embodiments, luminescent labels areidentified or distinguished based on luminescent intensity. In someembodiments, luminescent labels are identified or distinguished based onthe wavelength of the delivered excitation energy necessary to observean emitted photon. In some embodiments, luminescent labels areidentified or distinguished based on the wavelength of an emittedphoton. In some embodiments, luminescent labels are identified ordistinguished based on both luminescent lifetime and the wavelength ofthe delivered excitation energy necessary to observe an emitted photon.In some embodiments, luminescent labels are identified or distinguishedbased on both a luminescent intensity and the wavelength of thedelivered excitation energy necessary to observe an emitted photon. Insome embodiments, luminescent labels are identified or distinguishedbased on luminescent lifetime, luminescent intensity, and the wavelengthof the delivered excitation energy necessary to observe an emittedphoton. In some embodiments, luminescent labels are identified ordistinguished based on both luminescent lifetime and the wavelength ofan emitted photon. In some embodiments, luminescent labels areidentified or distinguished based on both a luminescent intensity andthe wavelength of an emitted photon. In some embodiments, luminescentlabels are identified or distinguished based on luminescent lifetime,luminescent intensity, and the wavelength of an emitted photon.

In certain embodiments, different types of molecules in a reactionmixture or experiment are labeled with different luminescent markers. Insome embodiments, the different markers have different luminescentproperties which can be distinguished. In some embodiments, thedifferent markers are distinguished by having different luminescentlifetimes, different luminescent intensities, different wavelengths ofemitted photons, or a combination thereof. The presence of a pluralityof types of molecules with different luminescent markers may allow fordifferent steps of a complex reaction to be monitored, or for differentcomponents of a complex reaction product to be identified. In someembodiments, the order in which the different types of molecules reactor interact can be determined.

In some embodiments, different nucleotides can be luminescently labeledwith a different number of the same luminescent molecule (e.g., one ormore of the same fluorescent dye). In some embodiments, changing thenumber of luminescent molecules in a luminescent label can allowdifferent nucleotides to be distinguished based on differentintensities. In some embodiments, different nucleotides can each belabeled with a different type of luminescent molecule and/or a differentnumber of each luminescent molecule. In some embodiments, differenttypes of luminescent molecules allow different nucleotides to bedistinguished based on different intensities and/or different lifetimes.

In certain embodiments, the luminescent properties of a plurality oftypes of molecules with different luminescent markers are used toidentify the sequence of a biomolecule, such as a nucleic acid orprotein. In some embodiments, the luminescent properties of a pluralityof types of molecules with different luminescent markers are used toidentify single molecules as they are incorporated during the synthesisof a biomolecule. In some embodiments, the luminescent properties of aplurality of types of nucleotides with different luminescent markers areused to identify single nucleotides as they are incorporated during asequencing reaction. This may allow for determination of the sequence ofa nucleic acid template. In some embodiments, the luminescently labelednucleotides are incorporated into a nucleic acid strand complementary tonucleic acid template. In some embodiments, the complementary strandcomprises a primer.

In certain embodiments, the plurality of types of nucleotides withdifferent luminescent markers comprises four types of luminescentlylabeled nucleotides. In some embodiments, the four luminescently labelednucleotides absorb and/or emit photons within one spectral range. Insome embodiments, three of the luminescently labeled nucleotides emitwithin a first spectral range, and a fourth luminescently labelednucleotide absorbs and/or emits photons within a second spectral range.In some embodiments, two of the luminescently labeled nucleotides emitwithin a first spectral range, and the a third and fourth luminescentlylabeled nucleotide emit luminescence within a second spectral range. Insome embodiments, two of the luminescently labeled nucleotides emitwithin a first spectral range, a third nucleotide ab absorbs and/oremits photons within a second spectral range, and a fourth nucleotideabsorbs and/or emits photons within a third spectral range. In someembodiments, each of the four luminescently labeled nucleotides absorbsand/or emits photons within a different spectral range. In someembodiments, each type of luminescently labeled nucleotide that absorbsand/or emits photons within the same spectral range has a differentluminescent lifetime or luminescent intensity, or both.

In some embodiments, four different types of nucleotides (e.g., adenine,guanine, cytosine, thymine/uracil) in a reaction mixture can each belabeled with one or more luminescent molecules. In some embodiments,each type of nucleotide can be connected to more than one of the sameluminescent molecule (e.g., two or more of the same fluorescent dyeconnected to a nucleotide). In some embodiments, each luminescentmolecule can be connected to more than one nucleotide (e.g., two or moreof the same nucleotide). In some embodiments, more than one nucleotidecan be connected to more than one luminescent molecule. In someembodiments, a protecting molecule serves as an anchor for attaching oneor more nucleotides (e.g., of the same type) and one or more luminescentmolecules (e.g., of the same type). In some embodiments, all fournucleotides are labeled with luminescent molecules that absorb and emitwithin the same spectral range (e.g., 520-570 nm).

In some embodiments, the luminescent labels among a set of fournucleotides can be selected from dyes comprising an aromatic orheteroaromatic compound and can be a pyrene, anthracene, naphthalene,acridine, stilbene, indole, benzindole, oxazole, carbazole, thiazole,benzothiazole, phenanthridine, phenoxazine, porphyrin, quinoline,ethidium, benzamide, cyanine, carbocyanine, salicylate, anthranilate,coumarin, fluoroscein, rhodamine, or other like compound. Exemplary dyesinclude xanthene dyes, such as fluorescein or rhodamine dyes,naphthalene dyes, coumarin dyes, acridine dyes, cyanine dyes,benzoxazole dyes, stilbene dyes, pyrene dyes, phthalocyanine dyes,phycobiliprotein dyes, squaraine dyes, and the like.

In some embodiments, the luminescent labels among a set of fournucleotides comprise Alexa Fluor® 546, Cy®3B, Alexa Fluor® 555 and AlexaFluor® 555, and the FRET pair Alexa Fluor® 555 and Cy®3.5. In someembodiments, the luminescent labels among a set of four nucleotidescomprise Alexa Fluor® 555, Cy®3.5, Alexa Fluor® 546, and DyLight®554-R1. In some embodiments, the luminescent labels among a set of fournucleotides comprise Alexa Fluor® 555, Cy®3.5, ATTO Rho6G, and DyLight®554-R1. In some embodiments, the luminescent labels among a set of fournucleotides comprise Alexa Fluor® 555, Cy®3B, ATTO Rho6G, and DyLight®554-R1. In some embodiments, the luminescent labels among a set of fournucleotides comprise Alexa Fluor® 555, Cy®3B, ATTO 542, and DyLight®554-R1. In some embodiments, the luminescent labels among a set of fournucleotides comprise Alexa Fluor® 555, Cy®3B, ATTO 542, and Alexa Fluor®546. In some embodiments, the luminescent labels among a set of fournucleotides comprise Cy®3.5, Cy®3B, ATTO Rho6G, and DyLight® 554-R1.

In certain embodiments, at least one type, at least two types, at leastthree types, or at least four of the types of luminescently labelednucleotides comprise a luminescent dye selected from the groupconsisting of 6-TAMRA, 5/6-Carboxyrhodamine 6G, Alexa Fluor® 546, AlexaFluor® 555, Alexa Fluor® 568, Alexa Fluor® 610, Alexa Fluor® 647,Aberrior Star 635, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514,ATTO 520, ATTO Rho6G, ATTO 542, ATTO 647N, ATTO Rho14, Chromis 630,Chromis 654A, Chromeo™ 642, CF™514, CF™532, CF™543, CF™546, CF™546,CF™555, CF™568, CF™633, CF™640R, CF™660C, CF™660R, CF™680R, Cy®3, Cy®3B,Cy®3.5, Cy®5, Cy®5.5, Dyomics-530, Dyomics-547P1, Dyomics-549P1,Dyomics-550, Dyomics-554, Dyomics-555, Dyomics-556, Dyomics-560,Dyomics-650, Dyomics-680, DyLight® 554-R1, DyLight® 530-R2, DyLight®594, DyLight® 635-B2, DyLight® 650, DyLight® 655-B4, DyLight® 675-B2,DyLight® 675-B4, DyLight® 680, HiLyte™ Fluor 532, HiLyte™ Fluor 555,HiLyte™ Fluor 594, LightCycler® 640R, Seta™ 555, Seta™ 670, Seta™700,Seta™u 647, and Seta™u 665, or are of formulae (Dye 101), (Dye 102),(Dye 103), (Dye 104), (Dye 105), or (Dye 106), as described herein.

In some embodiments, at least one type, at least two types, at leastthree types, or at least four of the types of luminescently labelednucleotides each comprise a luminescent dye selected from the groupconsisting of Alexa Fluor® 546, Alexa Fluor® 555, Cy®3B, Cy®3.5,DyLight® 554-R1, Alexa Fluor® 546, Atto Rho6G, ATTO 425, ATTO 465, ATTO488, ATTO 495, ATTO 514, ATTO 520, ATTO Rho6G, and ATTO 542.

In some embodiments, a first type of luminescently labeled nucleotidecomprises Alexa Fluor® 546, a second type of luminescently labelednucleotide comprises Cy®3B, a third type of luminescently labelednucleotide comprises two Alexa Fluor® 555, and a fourth type ofluminescently labeled nucleotide comprises Alexa Fluor® 555 and Cy®3.5.

In some embodiments, a first type of luminescently labeled nucleotidecomprises Alexa Fluor® 555, a second type of luminescently labelednucleotide comprises Cy®3.5, a third type of luminescently labelednucleotide comprises Alexa Fluor® 546, and a fourth type ofluminescently labeled nucleotide comprises DyLight® 554-R1.

In some embodiments, a first type of luminescently labeled nucleotidecomprises Alexa Fluor® 555, a second type of luminescently labelednucleotide comprises Cy®3.5, a third type of luminescently labelednucleotide comprises ATTO Rho6G, and a fourth type of luminescentlylabeled nucleotide comprises DyLight® 554-R1.

In some embodiments, a first type of luminescently labeled nucleotidecomprises Alexa Fluor® 555, a second type of luminescently labelednucleotide comprises Cy®3B, a third type of luminescently labelednucleotide comprises ATTO Rho6G, and a fourth type of luminescentlylabeled nucleotide comprises DyLight® 554-R1.

In some embodiments, a first type of luminescently labeled nucleotidecomprises Alexa Fluor® 555, a second type of luminescently labelednucleotide comprises Cy®3B, a third type of luminescently labelednucleotide comprises ATTO 542, and a fourth type of luminescentlylabeled nucleotide comprises DyLight® 554-R1.

In some embodiments, a first type of luminescently labeled nucleotidecomprises Alexa Fluor® 555, a second type of luminescently labelednucleotide comprises Cy®3B, a third type of luminescently labelednucleotide comprises ATTO 542, and a fourth type of luminescentlylabeled nucleotide comprises Alexa Fluor® 546.

In some embodiments, a first type of luminescently labeled nucleotidecomprises Cy®3.5, a second type of luminescently labeled nucleotidecomprises Cy®3B, a third type of luminescently labeled nucleotidecomprises ATTO Rho6G, and a fourth type of luminescently labelednucleotide comprises DyLight® 554-R1.

In some embodiments, at least one type, at least two types, at leastthree types, or at least four of the types of luminescently labelednucleotides comprise a luminescent dye selected from the groupconsisting of Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555,Alexa Fluor® 594, Alexa Fluor® 610, CF™532, CF™543, CF™555, CF™594,Cy®3, DyLight® 530-R2, DyLight® 554-R1, DyLight® 590-R2, DyLight® 594,DyLight® 610-B1, or are of formulae (Dye 101), (Dye 102), (Dye 103),(Dye 104), (Dye 105), or (Dye 106).

In some embodiments, a first and second type of luminescently labelednucleotide comprise a luminescent dye selected from the group consistingof Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, CF™532, CF™543,CF™555, Cy®3, DyLight® 530-R2, DyLight® 554-R1, and a third and fourthtype of luminescently labeled nucleotide comprise a luminescent dyeselected from the group consisting of Alexa Fluor® 594, Alexa Fluor®610, CF™594, DyLight® 590-R2, DyLight® 594, DyLight® 610-B1, or are offormulae (Dye 101), (Dye 102), (Dye 103), (Dye 104), (Dye 105), or (Dye106).

In certain embodiments, at least one type, at least two types, at leastthree types, or at least four of the types of luminescently-labelednucleotide molecules comprise a luminescent protein selected from thegroup consisting of TagBFP, mTagBFP2, Azurite, EBFP2, mKalamal, Sirius,Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2,monomeric Midoriishi-Cyan, TagCFP, mTFP1, EGFP, Emerald, SuperfolderGFP, monomeric Azami Green, TagGFP2, mUKG, mWasabi, Clover, mNeonGreen,EYFP, Citrine, Venus, SYFP2, TagYFP, monomeric Kusabira-Orange, mKOK,mKO2, mOrange, mOrange2, mRaspberry, mCherry, mStrawberry, mTangerine,tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mRuby2, mPlum, HcRed-Tandem,mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP, mKeima Red,LSS-mKate1, LSS-mKate2, mBeRFP, PA-GFP, PAmCherry1, PATagRFP, Kaede(green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, mEos2(green), mEos2 (red), PSmOrange, or Dronpa.

Aspects of the present application provide methods, and systems anddevices useful to such methods, for delivering an excitation energy to amolecule to be identified and detecting emitted photons after theexcitation. In certain embodiments, detecting comprises recording foreach detected luminescence the time duration between the luminescenceand the prior pulse of excitation energy. In certain embodiments,detecting comprises recording for each of a plurality of detectedluminescences the time duration between the luminescence and the priorpulse of excitation energy. In certain embodiments, a plurality ofpulses of excitation energy are delivered. The luminescent marker of themolecule to be identified may be excited by each pulse or a portion ofthe pulses. In certain embodiments, a plurality of luminescences aredetected by one or more sensors. The luminescent marker of the moleculeto be identified may emit luminescence after each excitation or aportion of the excitations. The fraction of excitation events thatresult in a luminescence is based on the luminescence quantum yield ofthe marker. In some embodiments, increasing the number of luminescentmolecules that comprise a luminescent marker can increase the quantumyield (e.g., increase the number of luminescence emissions).Additionally not all luminescences emitted by a marker will be detected,for example, some luminescences will be directed away from the sensors.In certain embodiments, the excitation energy or energies are selectedbased on the luminescent properties of the luminescent markers,including the absorption spectra and wavelengths at which a marker emitsphotons after excitation in a given spectral range.

In certain embodiments, the frequency of pulsed excitation energies isselected based on the luminescent properties (e.g., luminescentlifetime) of the luminescently labeled molecule to be detected. In someembodiments, the gap between pulses is longer than the luminescentlifetime of one or more luminescently labeled molecules being excited.In some embodiments, the gap is between about two times and about tentimes, between about ten times and about 100 times, or between about 100times and about 1000 times longer than the luminescent lifetime of oneor more luminescently labeled molecules being excited. In someembodiments, the gap is about 10 times longer than the luminescentlifetime of one or more luminescently labeled molecules being excited.

In certain embodiments, the frequency of pulsed excitation energies isselected based on the chemical process being monitored. For a sequencingreaction the number of pulses delivered to the target volume while aluminescently labeled nucleotide is being incorporated will in partdetermine the number of emitted photons detected. In some embodiments,the frequency is selected to allow for a sufficient number of photons tobe detected during the incorporation of a luminescently labelednucleotide, wherein a sufficient number is the number of photonsnecessary to distinguish the luminescently labeled nucleotide fromamongst a plurality of types of luminescently labeled nucleotides. Insome embodiments, the luminescently labeled nucleotide is distinguishedbased on the wavelength of the emitted photons. In some embodiments, theluminescently labeled nucleotide is distinguished based on theluminescent emission lifetime, e.g., the time between pulse excitationand emission detection. In some embodiments, the luminescently labelednucleotide is distinguished based on the wavelength and the luminescentemission lifetime of the emitted photons. In some embodiments, theluminescently labeled nucleotide is distinguished based on theluminescent intensity of the emission signal (e.g., based on thefrequency of emission or the total number of emission events within atime period). In some embodiments, the luminescently labeled nucleotideis distinguished based on the luminescent intensity of the emissionsignal and the luminescent lifetime. In some embodiments, theluminescently labeled nucleotide is distinguished based on theluminescent intensity and the wavelength. In some embodiments, theluminescently labeled nucleotide is distinguished based on theluminescent intensity, the wavelength, and the luminescent lifetime.

According to an aspect of the present application, a system comprisingan excitation source module and an integrated device is provided. Theexcitation source module comprises an excitation source configured toemit a pulse of excitation energy having a first duration of time. Theintegrated device includes a target volume configured to receive amolecule which, when coupled to the pulse of excitation energy emitsluminescence, a first energy path along which the pulse of excitationenergy moves from an energy source coupling component to the targetvolume, a sensor that detects the luminescence over a second duration oftime, wherein the second duration of time is greater than the firstduration of time, a second energy path along which the luminescencemoves from the target volume to the sensor, and a third energy pathalong which the pulse of excitation energy moves from the excitationsource to the energy source coupling component.

According to another aspect of the present application, an integrateddevice comprising a target volume and a sensor is provided. The targetvolume is configured to receive a sample labeled with one of a pluralityof luminescent markers, each of the plurality of luminescent markershaving a different lifetime value. The sensor is configured to detectluminescence from one of the plurality of luminescent markers over aplurality of time durations. The plurality of time durations areselected to differentiate among the plurality of luminescent markers.According to another aspect of the present application, an integrateddevice comprising a target volume and a plurality of sensors isprovided. The target volume is configured to receive a sample labeledwith one of a plurality of luminescent markers. Each of the plurality ofluminescent markers emit luminescence within one of a plurality ofspectral ranges and a portion of the plurality of luminescent markersthat emit luminescence at one of the plurality of spectral ranges eachhave different luminescent lifetimes. Each sensor of the plurality ofsensors is configured to detect one of the plurality of spectral rangesover a plurality of time durations and the plurality of time durationsare selected to differentiate among the portion of the plurality ofluminescent markers.

According to another aspect of the present application, a systemcomprising a plurality of excitation sources and an integrated device isprovided. The plurality of excitation sources emit a plurality ofexcitation energies. Each of the plurality of excitation sources emitpulses of one of the plurality of excitation energies. The integrateddevice includes a target volume configured to receive a sample labeledwith one of a plurality of luminescent markers and a sensor configuredto detect luminescence from one of the plurality of luminescent markersover a plurality of time durations after a pulse of one of the pluralityof excitation energies. A portion of the plurality of luminescentmarkers that emit luminescence after being illuminated by one of theplurality of excitation energies each have different lifetime values.The accumulation of a plurality of data timing the duration between apulse event and a luminescent emission differentiate among the pluralityof luminescent markers.

According to another aspect of the present application, a method ofsequencing a target nucleic acid is provided. In some embodiments, themethod of sequencing a target nucleic acid comprises steps of: (i)providing a mixture comprising (a) said target nucleic acid, (b) aprimer complementary to said target nucleic acid, (c) a nucleic acidpolymerase, and (d) nucleotides for incorporation into a growing nucleicacid strand complementary to said target nucleic acid, wherein saidnucleotides include different types of luminescently labelednucleotides, wherein said luminescently labeled nucleotides yielddetectable signals during sequential incorporation into said growingnucleic acid strand, which detectable signals for said different typesof luminescently labeled nucleotides are differentiable from one anotherin a time domain (e.g., by determining timing and/or frequency of thedetectable signals); (ii) subjecting said mixture of (i) to apolymerization reaction under conditions that are sufficient to yieldsaid growing nucleic acid strand by extension of said primer; (iii)measuring said detectable signals from said luminescently labelednucleotides during sequential incorporation into said growing nucleicacid strand; and (iv) determining the timing and/or frequency of saidmeasured detectable signals from said luminescently labeled nucleotidesupon sequential incorporation into said growing nucleic acid strand toidentify a time sequence of incorporation of said luminescently labelednucleotides into said growing nucleic acid strand, thereby determining asequence of said target nucleic acid.

In some embodiments, the target nucleic acid or the nucleic acidpolymerase is attached to a support. In some embodiments, the timesequence of incorporation is identified subsequent to subjecting themixture of (i) to the polymerization reaction. In some embodiments, thedetectable signals are optical signals. In some embodiments, the opticalsignals are luminescent signals. In some embodiments, determining thetiming and/or frequency of the measured detectable signals comprises (i)receiving said detectable signals at one or more sensors; and (ii)selectively directing charge carriers of a plurality of charge carriersproduced in response to said detectable signals received at said one ormore sensors into at least one charge carrier storage region based upontimes at which said charge carriers are produced.

In some embodiments, the timing and/or frequency of said measureddetectable signals comprise measurements of decay lifetimes. In someembodiments, the timing and/or frequency of the measured detectablesignals comprise measurements of arrival times of the detectable signalsat one or more sensors that detect the detectable signals. In someembodiments, the method further comprises segregating charge carriersproduced by the detectable signals into bins associated with the one ormore sensors based on the arrival times of the detectable signals. Insome embodiments, the timing and/or frequency of the measured detectablesignals are non-spectral measurements.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the figures, described herein,are for illustration purposes only. It is to be understood that in someinstances various aspects of the invention may be shown exaggerated orenlarged to facilitate an understanding of the invention. In thedrawings, like reference characters generally refer to like features,functionally similar and/or structurally similar elements throughout thevarious figures. The drawings are not necessarily to scale, emphasisinstead being placed upon illustrating the principles of the teachings.The drawings are not intended to limit the scope of the presentteachings in any way.

The features and advantages of the present invention will become moreapparent from the detailed description set forth below when taken inconjunction with the drawings.

When describing embodiments in reference to the drawings, directionreferences (“above,” “below,” “top,” “bottom,” “left,” “right,”“horizontal,” “vertical,” etc.) may be used. Such references areintended merely as an aid to the reader viewing the drawings in a normalorientation. These directional references are not intended to describe apreferred or only orientation of an embodied device. A device may beembodied in other orientations.

As is apparent from the detailed description, the examples depicted inthe figures (e.g., FIGS. 1-37) and further described for the purpose ofillustration throughout the application describe non-limitingembodiments, and in some cases may simplify certain processes or omitfeatures or steps for the purpose of clearer illustration.

FIG. 1 shows a non-limiting schematic of a sample well (e.g., a samplewell) containing various components for nucleic acid sequencing.

FIG. 2 shows a non-limiting, exemplary experiment of nucleic acidsequencing for four stages; (A) before incorporation of a luminescentlylabeled nucleotide; (B) a first incorporation event; (C) a periodbetween the first and second incorporation events; and (D) a secondincorporation event; along with corresponding examples of raw andprocessed data during stages (A)-(D).

FIG. 3 depicts a non-limiting signal vs. emission time for fourluminescent molecules with different luminescent lifetimes andnormalized cumulative distribution function for the probability ofdecay.

FIG. 4 shows a non-limiting chart of luminescent lifetimes and a chartfor luminescent intensities for exemplary luminescently labelednucleotides.

FIG. 5 depicts a non-limiting sequencing experiment with fourluminescently labeled nucleotides: (A) trace of detected luminescencesfrom green and red pulses; (B) reduction of data from green pulses basedon luminescent lifetime and intensity of each nucleotide incorporation;and (C) alignment of the experimentally determined sequence with thetemplate sequence.

FIG. 6 depicts a non-limiting sequencing experiment with fourluminescently labeled nucleotides: (A) trace of detected luminescencesfrom green pulses; (B) reduction of data from green pulses based onluminescent lifetime and intensity of each nucleotide incorporation; and(C) alignment of the experimentally determined sequence with thetemplate sequence.

FIG. 7 shows a non-limiting, exemplary process for surface preparation,including the steps of (a) deposition, (b) passivation, (c)silinazation, (d) complex loading, and (e) sequencing reactioninitiation.

FIG. 8 shows two non-limiting methods for passivation of metal oxidesurfaces with phosphonate-PEG groups.

FIG. 9 shows two non-limiting methods for functionalization of glasswith of silane-PEG-biotin and silane-PEG mixtures.

FIG. 10 depicts non-limiting examples of one or more luminescentmolecules and one or more nucleotides connected via a protectingmolecule.

FIG. 11 depicts non-limiting examples of configurations involving one ormore nucleotides and one or more luminescent molecules attached viasplit linkers to a protecting molecule.

FIG. 12 depicts a non-limiting embodiment of configurations ofluminescently-labeled nucleotide molecules that can be used in asequencing reaction and an exemplary sequencing experiment, wherein theluminescent properties of a luminescently-labeled nucleotide are used toidentify the base being incorporated into the sequencing reaction.

FIG. 13 depicts a non-limiting example of engineering non-functionalbinding sites into a protecting molecule to attach luminescent moleculesand nucleotides at selected sites.

FIG. 14 depicts a non-limiting example in which a luminescent moleculeand a nucleotide are connected via a protecting molecule, wherein thelinkers attaching the luminescent molecule and the nucleotide to theprotecting molecule comprise oligonucleotides.

FIG. 15 depicts a non-limiting example in which a luminescent moleculeis attached to a protecting molecule via annealed complementaryoligonucleotides, wherein each oligonucleotide strand contains onenon-covalent binding ligand compatible with a binding site on theprotecting molecule.

FIG. 16 depicts a non-limiting example of utilizing genetically encodedtags to attach a luminescent molecule and a nucleotide at independentsites of a protecting molecule.

FIG. 17 depicts a non-limiting example of utilizing reactive groups atthe terminal groups of a protein to attach a luminescent molecule and anucleotide at independent sites of a protecting molecule.

FIG. 18 depicts a non-limiting example of utilizing unnatural aminoacids to attach a luminescent molecule and a nucleotide at independentsites of a protecting molecule.

FIG. 19A and FIG. 19B depict non-limiting examples of a luminescentmolecule and a nucleotide separated by a non-protein protectingmolecule.

FIG. 20 depicts a non-limiting embodiment of a protecting moleculecomprised of a protein-protein binding pair.

FIG. 21 depicts non-limiting examples of linker configurations.

FIG. 22 depicts a non-limiting reaction scheme for nucleotide linkersynthesis and exemplary structures.

FIG. 23A is a block diagram representation of an apparatus that may beused for rapid, mobile analysis of biological and chemical specimens,according to some embodiments.

FIG. 23B is a block diagram of an integrated device and an instrument,according to some embodiments.

FIG. 24A depicts a row of pixels of an integrated device, according tosome embodiments.

FIG. 24B depicts excitation energy coupling to sample wells in a row ofpixels and emission energy from each sample well directed towardssensors, according to some embodiments.

FIG. 25 depicts an integrated device and an excitation source, accordingto some embodiments.

FIG. 26 depicts an integrated device and an excitation source, accordingto some embodiments.

FIG. 27A depicts a sample well formed in a pixel region of an integrateddevice, according to one embodiment.

FIG. 27B depicts excitation energy incident on a sample well, accordingto some embodiments.

FIG. 27C depicts a sample well that includes a divot, which increasesexcitation energy at an excitation region associated with the samplewell in some embodiments.

FIG. 28 depicts a sample well and divot, according to some embodiments.

FIGS. 29A and 29B depict a sensor with time bins, according to someembodiments.

FIG. 30A depicts an exemplary system for providing light pulses,according to some embodiments.

FIG. 30B depicts a plot of light intensity as a function of time.

FIG. 31A depicts a schematic for performing measurements, according tosome embodiments.

FIG. 31B depicts a plot of light signal as a function of time, accordingto some embodiments.

FIG. 31C depicts a signal profile for markers across time bins,according to some embodiments.

FIG. 32A depicts a plot of light signal as a function of time, accordingto some embodiments.

FIG. 32B depicts a signal profile for markers across time bins,according to some embodiments.

FIG. 33A depicts a schematic for performing measurements, according tosome embodiments.

FIG. 33B depicts a plot of lifetime as a function of emissionwavelength, according to some embodiments.

FIG. 34A depicts a plot of light signal as a function of wavelength,according to some embodiments.

FIG. 34B depicts a plot of light signal as a function of time, accordingto some embodiments.

FIG. 35A depicts a signal profile for markers across time bins formultiple sensors, according to some embodiments.

FIG. 35B depicts a plot of light signal as a function of time, accordingto some embodiments.

FIG. 35C depicts a signal profile for a marker across time bins formultiple sensors, according to some embodiments.

FIG. 36A depicts a schematic for performing measurements, according tosome embodiments.

FIG. 36B depicts a plot of light signal as a function of wavelength,according to some embodiments.

FIG. 37A depicts a plot of light signal as a function of time, accordingto some embodiments.

FIG. 37B depicts a signal profile for a marker across time bins formultiple sensors, according to some embodiments.

DETAILED DESCRIPTION

The inventors have developed new methods, compositions, and devices foridentifying single molecules based on one or more luminescent propertiesof those molecules. In some embodiments, a molecule is identified basedon its luminescent lifetime, absorption spectra, emission spectra,luminescent quantum yield, luminescent intensity, or a combination oftwo or more thereof. Identifying may mean assigning the exact molecularidentity of a molecule, or may mean distinguishing or differentiatingthe particular molecule from a set of possible molecules. In someembodiments, a plurality of single molecules can be distinguished fromeach other based on different luminescent lifetimes, absorption spectra,emission spectra, luminescent quantum yields, luminescent intensities,or combinations of two or more thereof. In some embodiments, a singlemolecule is identified (e.g., distinguished from other molecules) byexposing the molecule to a series of separate light pulses andevaluating the timing or other properties of each photon that is emittedfrom the molecule. In some embodiments, information for a plurality ofphotons emitted sequentially from a single molecule is aggregated andevaluated to identify the molecule. In some embodiments, a luminescentlifetime of a molecule is determined from a plurality of photons thatare emitted sequentially from the molecule, and the luminescent lifetimecan be used to identify the molecule. In some embodiments, a luminescentintensity of a molecule is determined from a plurality of photons thatare emitted sequentially from the molecule, and the luminescentintensity can be used to identify the molecule. In some embodiments, aluminescent lifetime and luminescent intensity of a molecule isdetermined from a plurality of photons that are emitted sequentiallyfrom the molecule, and the luminescent lifetime and luminescentintensity can be used to identify the molecule.

Aspects of the present application are useful for detecting and/oridentifying one or more biological or chemical molecules. In someembodiments, chemical or biological reactions can be evaluated bydetermining the presence or absence of one or more reagents or productsat one or more time points.

Aspects of the present application interrogate a molecule by exposingthe molecule to light and determining one or more properties of one ormore photons emitted from the molecule. In certain embodiments, themolecule is interrogated by exposing the molecule to a pulse of lightand determining one or more properties of a photon emitted from themolecule. In some embodiments, the molecule is exposed to a plurality ofseparate light pulse events and one or more properties of separatephotons emitted after separate light pulse events are determined. Insome embodiments, the molecule does not emit a photon in response toeach light pulse. However, a plurality of emitted photons can beevaluated by exposing the molecule to a series of separate light pulsesand evaluating separate photons that are emitted after a subset of thelight pulse events (e.g., photons emitted after about 10% of pulseevents, or photons emitted after about 1% of pulse events).

Aspects of the present application are useful to monitor a chemical orbiological reaction by determining the presence or absence of one ormore reagents, intermediates, and/or products of the reaction at one ormore time points. In some embodiments, the progression of a reactionover time can be analyzed by exposing a reaction sample to a series ofseparate light pulses and analyzing any emitted photon that is detectedafter each light pulse.

Accordingly, in some aspects of the application, a reaction sample isexposed to a plurality of separate light pulses and a series of emittedphotons are detected and analyzed. In some embodiments, the series ofemitted photons provides information about a single molecule that ispresent and that does not change in the reaction sample over the time ofthe experiment. However, in some embodiments, the series of emittedphotons provides information about a series of different molecules thatare present at different times in the reaction sample (e.g., as areaction or process progresses).

In some embodiments, aspects of the present application can be used toassay biological samples, for example to determine the sequence of oneor more nucleic acids or polypeptides in the sample and/or to determinethe presence or absence of one or more nucleic acid or polypeptidevariants (e.g., one or more mutations in a gene of interest) in thesample. In some embodiments, tests can be performed on patient samples(e.g., human patient samples) to provide nucleic acid sequenceinformation or to determine the presence or absence of one or morenucleic acids of interest for diagnostic, prognostic, and/or therapeuticpurposes. In some examples, diagnostic tests can include sequencing anucleic acid molecule in a biological sample of a subject, for exampleby sequencing cell free deoxyribonucleic acid (DNA) molecules and/orexpression products (e.g., ribonucleic acid (RNA)) in a biologicalsample of the subject.

In some embodiments, one or more molecules that are being analyzed(e.g., interrogated and/or identified) using luminescent lifetime and/orintensity can be labeled molecules (e.g., molecules that have beenlabeled with one or more luminescent markers). In some embodiments,individual subunits of biomolecules may be identified using markers. Insome examples, luminescent markers are used to identify individualsubunits of biomolecules. Some embodiments use luminescent markers (alsoreferred to herein as “markers”), which may be exogenous or endogenousmarkers. Exogenous markers may be external luminescent markers used as areporter and/or tag for luminescent labeling. Examples of exogenousmarkers may include, but are not limited to, fluorescent molecules,fluorophores, fluorescent dyes, fluorescent stains, organic dyes,fluorescent proteins, species that participate in fluorescence resonanceenergy transfer (FRET), enzymes, and/or quantum dots. Other exogenousmarkers are known in the art. Such exogenous markers may be conjugatedto a probe or functional group (e.g., molecule, ion, and/or ligand) thatspecifically binds to a particular target or component. Attaching anexogenous tag or reporter to a probe allows identification of the targetthrough detection of the presence of the exogenous tag or reporter.Examples of probes may include proteins, nucleic acid (e.g., DNA, RNA)molecules, lipids and antibody probes. The combination of an exogenousmarker and a functional group may form any suitable probes, tags, and/orlabels used for detection, including molecular probes, labeled probes,hybridization probes, antibody probes, protein probes (e.g.,biotin-binding probes), enzyme labels, fluorescent probes, fluorescenttags, and/or enzyme reporters.

Although the present disclosure makes reference to luminescent markers,other types of markers may be used with devices, systems and methodsprovided herein. Such markers may be mass tags, electrostatic tags,electrochemical labels, or any combination thereof.

While exogenous markers may be added to a sample, endogenous markers maybe already part of the sample. Endogenous markers may include anyluminescent marker present that may luminesce or “autofluoresce” in thepresence of excitation energy. Autofluorescence of endogenousfluorophores may provide for label-free and noninvasive labeling withoutrequiring the introduction of exogenous fluorophores. Examples of suchendogenous fluorophores may include hemoglobin, oxyhemoglobin, lipids,collagen and elastin crosslinks, reduced nicotinamide adeninedinucleotide (NADH), oxidized flavins (FAD and FMN), lipofuscin,keratin, and/or porphyrins, by way of example and not limitation.

Having recognized the need for simple, less complex apparatuses forperforming single molecule detection and/or nucleic acid sequencing, theinventors have conceived of techniques for detecting single moleculesusing sets of luminescent tags (e.g., luminescent markers, luminescentlabels) to label different molecules. Such single molecules may benucleotides or amino acids having tags. Tags may be detected while boundto single molecules, upon release from the single molecules, or whilebound to and upon release from the single molecules. In some examples,tags are luminescent tags. Each luminescent tag in a selected set isassociated with a respective molecule. For example, a set of four tagsmay be used to “label” the nucleobases present in DNA—each tag of theset being associated with a different nucleobase, e.g., a first tagbeing associated with adenine (A), a second tag being associated withcytosine (C), a third tag being associated with guanine (G), and afourth tag being associated with thymine (T). Moreover, each of theluminescent tags in the set of tags has different properties that may beused to distinguish a first tag of the set from the other tags in theset. In this way, each tag is uniquely identifiable using one or more ofthese distinguishing characteristics. By way of example and notlimitation, the characteristics of the tags that may be used todistinguish one tag from another may include the emission energy and/orwavelength of the light that is emitted by the tag in response toexcitation energy, the wavelength of the excitation light that isabsorbed by a particular tag to place the tag in an excited state,and/or the emission lifetime of the tag.

Sequencing

Some aspects of the application are useful for sequencing biologicalpolymers, such as nucleic acids and proteins. In some embodiments,methods, compositions, and devices described in the application can beused to identify a series of nucleotide or amino acid monomers that areincorporated into a nucleic acid or protein (e.g., by detecting atime-course of incorporation of a series of labeled nucleotide or aminoacid monomers). In some embodiments, methods, compositions, and devicesdescribed in the application can be used to identify a series ofnucleotides that are incorporated into a template-dependent nucleic acidsequencing reaction product synthesized by a polymerase enzyme.

In certain embodiments, the template-dependent nucleic acid sequencingproduct is carried out by naturally occurring nucleic acid polymerases.In some embodiments, the polymerase is a mutant or modified variant of anaturally occurring polymerase. In some embodiments, thetemplate-dependent nucleic acid sequence product will comprise one ormore nucleotide segments complementary to the template nucleic acidstrand. In one aspect, the application provides a method of determiningthe sequence of a template (or target) nucleic acid strand bydetermining the sequence of its complementary nucleic acid strand.

In another aspect, the application provides methods of sequencing targetnucleic acids by sequencing a plurality of nucleic acid fragments,wherein the target nucleic acid comprises the fragments. In certainembodiments, the method comprises combining a plurality of fragmentsequences to provide a sequence or partial sequence for the parenttarget nucleic acid. In some embodiments, the step of combining isperformed by computer hardware and software. The methods describedherein may allow for a set of related target nucleic acids, such as anentire chromosome or genome to be sequenced.

During sequencing, a polymerizing enzyme may couple (e.g., attach) to apriming location of a target nucleic acid molecule. The priming locationcan be a primer that is complementary to a portion of the target nucleicacid molecule. As an alternative the priming location is a gap or nickthat is provided within a double stranded segment of the target nucleicacid molecule. A gap or nick can be from 0 to at least 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 20, 30, or 40 nucleotides in length. A nick can provide abreak in one strand of a double stranded sequence, which can provide apriming location for a polymerizing enzyme, such as, for example, astrand displacing polymerase enzyme.

In some cases, a sequencing primer can be annealed to a target nucleicacid molecule that may or may not be immobilized to a solid support. Asolid support can comprise, for example, a sample well (e.g., ananoaperture, a reaction chamber) on a chip used for nucleic acidsequencing. In some embodiments, a sequencing primer may be immobilizedto a solid support and hybridization of the target nucleic acid moleculealso immobilizes the target nucleic acid molecule to the solid support.In some embodiments, a polymerase is immobilized to a solid support andsoluble primer and target nucleic acid are contacted to the polymerase.However, in some embodiments a complex comprising a polymerase, a targetnucleic acid and a primer is formed in solution and the complex isimmobilized to a solid support (e.g., via immobilization of thepolymerase, primer, and/or target nucleic acid). In some embodiments,none of the components in a sample well (e.g., a nanoaperture, areaction chamber) are immobilized to a solid support. For example, insome embodiments, a complex comprising a polymerase, a target nucleicacid, and a primer is formed in solution and the complex is notimmobilized to a solid support.

Under appropriate conditions, a polymerase enzyme that is contacted toan annealed primer/target nucleic acid can add or incorporate one ormore nucleotides onto the primer, and nucleotides can be added to theprimer in a 5′ to 3′, template-dependent fashion. Such incorporation ofnucleotides onto a primer (e.g., via the action of a polymerase) cangenerally be referred to as a primer extension reaction. Each nucleotidecan be associated with a detectable tag that can be detected andidentified (e.g., based on its luminescent lifetime and/or othercharacteristics) during the nucleic acid extension reaction and used todetermine each nucleotide incorporated into the extended primer and,thus, a sequence of the newly synthesized nucleic acid molecule. Viasequence complementarity of the newly synthesized nucleic acid molecule,the sequence of the target nucleic acid molecule can also be determined.In some cases, annealing of a sequencing primer to a target nucleic acidmolecule and incorporation of nucleotides to the sequencing primer canoccur at similar reaction conditions (e.g., the same or similar reactiontemperature) or at differing reaction conditions (e.g., differentreaction temperatures). In some embodiments, sequencing by synthesismethods can include the presence of a population of target nucleic acidmolecules (e.g., copies of a target nucleic acid) and/or a step ofamplification of the target nucleic acid to achieve a population oftarget nucleic acids. However, in some embodiments sequencing bysynthesis is used to determine the sequence of a single molecule in eachreaction that is being evaluated (and nucleic acid amplification is notrequired to prepare the target template for sequencing). In someembodiments, a plurality of single molecule sequencing reactions areperformed in parallel (e.g., on a single chip) according to aspects ofthe present application. For example, in some embodiments, a pluralityof single molecule sequencing reactions are each performed in separatereaction chambers (e.g., nanoapertures, sample wells) on a single chip.

Embodiments are capable of sequencing single nucleic acid molecules withhigh accuracy and long read lengths, such as an accuracy of at leastabout 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%,99.99%, 99.999%, or 99.9999%, and/or read lengths greater than or equalto about 10 base pairs (bp), 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500bp, 1000 bp, 10,000 bp, 20,000 bp, 30,000 bp, 40,000 bp, 50,000 bp, or100,000 bp. In some embodiments, the target nucleic acid molecule usedin single molecule sequencing is a single stranded target nucleic acid(e.g., deoxyribonucleic acid (DNA), DNA derivatives, ribonucleic acid(RNA), RNA derivatives) template that is added or immobilized to asample well (e.g., nanoaperture) containing at least one additionalcomponent of a sequencing reaction (e.g., a polymerase such as, a DNApolymerase, a sequencing primer) immobilized or attached to a solidsupport such as the bottom or side walls of the sample well. The targetnucleic acid molecule or the polymerase can be attached to a samplewall, such as at the bottom or side walls of the sample well directly orthrough a linker. The sample well (e.g., nanoaperture) also can containany other reagents needed for nucleic acid synthesis via a primerextension reaction, such as, for example suitable buffers, co-factors,enzymes (e.g., a polymerase) and deoxyribonucleoside polyphosphates,such as, e.g., deoxyribonucleoside triphosphates, includingdeoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP),deoxyguanosine triphosphate (dGTP), deoxyuridine triphosphate (dUTP) anddeoxythymidine triphosphate (dTTP) dNTPs, that include luminescent tags,such as fluorophores. In some embodiments, each class of dNTPs (e.g.,adenine-containing dNTPs (e.g., dATP), cytosine-containing dNTPs (e.g.,dCTP), guanine-containing dNTPs (e.g., dGTP), uracil-containing dNTPs(e.g., dUTPs) and thymine-containing dNTPs (e.g., dTTP)) is conjugatedto a distinct luminescent tag such that detection of light emitted fromthe tag indicates the identity of the dNTP that was incorporated intothe newly synthesized nucleic acid. Emitted light from the luminescenttag can be detected and attributed to its appropriate luminescent tag(and, thus, associated dNTP) via any suitable device and/or method,including such devices and methods for detection described elsewhereherein. The luminescent tag may be conjugated to the dNTP at anyposition such that the presence of the luminescent tag does not inhibitthe incorporation of the dNTP into the newly synthesized nucleic acidstrand or the activity of the polymerase. In some embodiments, theluminescent tag is conjugated to the terminal phosphate (e.g., the gammaphosphate) of the dNTP.

In some embodiments, the single-stranded target nucleic acid templatecan be contacted with a sequencing primer, dNTPs, polymerase and otherreagents necessary for nucleic acid synthesis. In some embodiments, allappropriate dNTPs can be contacted with the single-stranded targetnucleic acid template simultaneously (e.g., all dNTPs are simultaneouslypresent) such that incorporation of dNTPs can occur continuously. Inother embodiments, the dNTPs can be contacted with the single-strandedtarget nucleic acid template sequentially, where the single-strandedtarget nucleic acid template is contacted with each appropriate dNTPseparately, with washing steps in between contact of the single-strandedtarget nucleic acid template with differing dNTPs. Such a cycle ofcontacting the single-stranded target nucleic acid template with eachdNTP separately followed by washing can be repeated for each successivebase position of the single-stranded target nucleic acid template to beidentified.

In some embodiments, the sequencing primer anneals to thesingle-stranded target nucleic acid template and the polymeraseconsecutively incorporates the dNTPs (or other deoxyribonucleosidepolyphosphate) to the primer based on the single-stranded target nucleicacid template. The unique luminescent tag associated with eachincorporated dNTP can be excited with the appropriate excitation lightduring or after incorporation of the dNTP to the primer and its emissioncan be subsequently detected, using, any suitable device(s) and/ormethod(s), including devices and methods for detection describedelsewhere herein. Detection of a particular emission of light (e.g.,having a particular emission lifetime, intensity, spectrum and/orcombination thereof) can be attributed to a particular dNTPincorporated. The sequence obtained from the collection of detectedluminescent tags can then be used to determine the sequence of thesingle-stranded target nucleic acid template via sequencecomplementarity.

While the present disclosure makes reference to dNTPs, devices, systemsand methods provided herein may be used with various types ofnucleotides, such as ribonucleotides and deoxyribonucleotides (e.g.,deoxyribonucleoside polyphosphates with at least 4, 5, 6, 7, 8, 9, or 10phosphate groups). Such ribonucleotides and deoxyribonucleotides caninclude various types of tags (or markers) and linkers.

Example of Nucleic Acid Sequencing

The following example is meant to illustrate some of the methods,compositions and devices described herein. All aspects of the exampleare non-limiting. FIG. 1 schematically illustrates the setup of a singlemolecule nucleic acid sequencing method. 1-110 is a sample well (e.g.,nanoaperture, reaction chamber) configured to contain a single complexcomprising a nucleic acid polymerase 1-101, a target nucleic acid 1-102to be sequenced, and a primer 1-104. In this example, a bottom region ofsample well 1-110 is depicted as a target volume (e.g., the excitationregion) 1-120.

As described elsewhere herein, the target volume is a volume towardswhich the excitation energy is directed. In some embodiments, the volumeis a property of both the sample well volume and the coupling ofexcitation energy to the sample well. The target volume may beconfigured to limit the number of molecules or complexes confined in thetarget volume. In some embodiments, the target volume is configured toconfine a single molecule or complex. In some embodiments, the targetvolume is configured to confine a single polymerase complex. In FIG. 1the complex comprising polymerase 1-101 is confined in target volume1-120. The complex may optionally be immobilized by attachment to asurface of the sample well. Exemplary processes for sample well surfacepreparation and functionalization are depicted in FIGS. 7-9 discussed infurther detail elsewhere in the application. In this example the complexis immobilized by a linker 1-103 comprising one or more biomolecules(e.g., biotin) suitable for attaching the linker to the polymerase1-101.

The volume of the aperture also contains a reaction mixture withsuitable solvent, buffers, and other additives necessary for thepolymerase complex to synthesize a nucleic acid strand. The reactionmixture also contains a plurality of types of luminescently labelednucleotides. Each type of nucleotide is represented by the symbols *-A,@-T, $-G, #-C, wherein A, T, G, and C represent the nucleotide base, andthe symbols *, @, $, and # represent a unique luminescent label attachedto each nucleotide, through linker -. In FIG. 1, a #-C nucleotide iscurrently being incorporated into the complementary strand 1-102. Theincorporated nucleotide is within the target volume 1-120.

FIG. 1 also indicates with arrows the concept of an excitation energybeing delivered to a vicinity of the target volume, and a luminescencebeing emitted towards a detector. The arrows are schematic, and are notmeant to indicate the particular orientation of excitation energydelivery or luminescence. In some embodiments, the excitation energy isa pulse of light from a light source. The excitation energy may travelthrough one or more device components, such as waveguides or filters,between the light source and the vicinity of the target volume. Theemission energy may also travel through one or more device components,such as waveguides or filters, between the luminescent molecule and thedetector. Some luminescences may emit on a vector which is not directedto the detector (e.g., towards the sidewall of the sample well) and maynot be detected.

FIG. 2 schematically illustrates a sequencing process in a single samplewell (e.g., a nanoaperture) over time. Stages A through D depict asample well with a polymerase complex as in FIG. 1. Stage A depicts theinitial state before any nucleotides have been added to the primer.Stage B depicts the incorporation event of a luminescently labelednucleotide (#-C). Stage C depicts the period between incorporationevents. In this example, nucleotide C has been added to the primer, andthe label and linker previously attached to the luminescently labelednucleotide (#-C) has been cleaved. Stage D depicts a secondincorporation event of a luminescently labeled nucleotide (*-A). Thecomplementary strand after Stage D consists of the primer, a Cnucleotide, and an A nucleotide.

Stage A and C, both depict the periods before or between incorporationevents, which are indicated in this example to last for about 10milliseconds. In stages A and C, because there is no nucleotide beingincorporated, there is no luminescently labeled nucleotide in the targetvolume (not drawn in FIG. 2), though background luminescence or spuriousluminescence from luminescently labeled nucleotide which is not beingincorporated may be detected. Stage B and D show incorporation events ofdifferent nucleotides (#-C, and *-A, respectively). In this examplethese events are also indicated to last for about 10 milliseconds.

The row labeled “Raw bin data” depicts the data generated during eachStage. Throughout the example experiment, a plurality of pulses of lightare delivered to the vicinity of the target volume. For each pulse adetector is configured to record any emitted photon received by thedetector. When an emitted photon is received by the detector it isseparated into one of a plurality of time bins, of which there are 3 inthis example. In some embodiments, the detector is configured withbetween 2 and 16 time bins. The “Raw bin data” records a value of 1(shortest bars), 2 (medium bars), or 3 (longest bars), corresponding tothe shortest, middle, and longest bins, respectively. Each bar indicatesdetection of an emitted photon.

Since there is no luminescently labeled nucleotide present in the targetvolume for Stage A or C, there are no photons detected. For each ofStage B and D a plurality of photon emission events (luminescent eventsor “luminescences” as used herein) is detected during the incorporationevent. Luminescent label # has a shorter luminescence lifetime thanluminescent label *. The Stage B data is thus depicted as havingrecorded lower average bin values, than Stage D where the bin values arehigher.

The row labeled “Processed data” depicts raw data which has beenprocessed to indicate the number (counts) of emitted photons at timesrelative to each pulse. Since each bar corresponds to the photon countof a particular time bin, the exemplary curves depicting processed datacorrespond to raw bin data comprising more time bins than the three timebins described in the figure. In this example, the data is onlyprocessed to determine luminescent lifetime, but the data may also beevaluated for other luminescent properties, such as luminescentintensity or the wavelength of the absorbed or emitted photons. Theexemplary processed data approximates an exponential decay curvecharacteristic for the luminescence lifetime of the luminescent label inthe target volume. Because luminescent label # has a shorterluminescence lifetime than luminescent label *, the processed data forStage B has fewer counts at longer time durations, while the processeddata for Stage D has relatively more counts at longer time durations.

The example experiment of FIG. 2 would identify the first twonucleotides added to the complementary strand as CA. For DNA, thesequence of the target strand immediately after the region annealed tothe primer would thus be identified as GT. In this example thenucleotides C and A could be distinguished from amongst the plurality ofC, G, T, and A, based on luminescent lifetime alone. In someembodiments, other properties, such as the luminescent intensity or thewavelength of the absorbed or emitted photons may be necessary todistinguish one or more particular nucleotide.

Signals emitted upon the incorporation of nucleotides can be stored inmemory and processed at a later point in time to determine the sequenceof the target nucleic acid template. This may include comparing thesignals to a reference signals to determine the identities of theincorporated nucleotides as a function of time. Alternatively or inaddition to, signal emitted upon the incorporation of nucleotide can becollected and processed in real time (e.g., upon nucleotideincorporation) to determine the sequence of the target nucleic acidtemplate in real time.

The term “nucleic acid,” as used herein, generally refers to a moleculecomprising one or more nucleic acid subunits. A nucleic acid may includeone or more subunits selected from adenine (A), cytosine (C), guanine(G), thymine (T), and uracil (U), or variants thereof. In some examples,a nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA),or derivatives thereof. A nucleic acid may be single-stranded or doublestranded. A nucleic acid may be circular.

The term “nucleotide,” as used herein, generally refers to a nucleicacid subunit, which can include A, C, G, T or U, or variants or analogsthereof. A nucleotide can include any subunit that can be incorporatedinto a growing nucleic acid strand. Such subunit can be an A, C, G, T,or U, or any other subunit that is specific to one or more complementaryA, C, G, T or U, or complementary to a purine (e.g., A or G, or variantor analogs thereof) or a pyrimidine (e.g., C, T or U, or variant oranalogs thereof). A subunit can enable individual nucleic acid bases orgroups of bases (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA, oruracil-counterparts thereof) to be resolved.

A nucleotide generally includes a nucleoside and at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, or more phosphate (PO₃) groups. A nucleotide can includea nucleobase, a five-carbon sugar (either ribose or deoxyribose), andone or more phosphate groups. Ribonucleotides are nucleotides in whichthe sugar is ribose. Deoxyribonucleotides are nucleotides in which thesugar is deoxyribose. A nucleotide can be a nucleoside monophosphate ora nucleoside polyphosphate. A nucleotide can be a deoxyribonucleosidepolyphosphate, such as, e.g., a deoxyribonucleoside triphosphate, whichcan be selected from deoxyadenosine triphosphate (dATP), deoxycytidinetriphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyuridinetriphosphate (dUTP) and deoxythymidine triphosphate (dTTP) dNTPs, thatinclude detectable tags, such as luminescent tags or markers (e.g.,fluorophores).

A nucleoside polyphosphate can have ‘n’ phosphate groups, where ‘n’ is anumber that is greater than or equal to 2, 3, 4, 5, 6, 7, 8, 9, or 10.Examples of nucleoside polyphosphates include nucleoside diphosphate andnucleoside triphosphate. A nucleotide can be a terminal phosphatelabeled nucleoside, such as a terminal phosphate labeled nucleosidepolyphosphate. Such label can be a luminescent (e.g., fluorescent orchemiluminescent) label, a fluorogenic label, a colored label, achromogenic label, a mass tag, an electrostatic label, or anelectrochemical label. A label (or marker) can be coupled to a terminalphosphate through a linker. The linker can include, for example, atleast one or a plurality of hydroxyl groups, sulfhydryl groups, aminogroups or haloalkyl groups, which may be suitable for forming, forexample, a phosphate ester, a thioester, a phosphoramidate or an alkylphosphonate linkage at the terminal phosphate of a natural or modifiednucleotide. A linker can be cleavable so as to separate a label from theterminal phosphate, such as with the aid of a polymerization enzyme.Examples of nucleotides and linkers are provided in U.S. Pat. No.7,041,812, which is entirely incorporated herein by reference.

A nucleotide (e.g., a nucleotide polyphosphate) can comprise amethylated nucleobase. For example, a methylated nucleotide can be anucleotide that comprises one or more methyl groups attached to thenucleobase (e.g., attached directly to a ring of the nucleobase,attached to a substituent of a ring of the nucleobase). Exemplarymethylated nucleobases include 1-methylthymine, 1-methyluracil,3-methyluracil, 3-methylcytosine, 5-methylcytosine, 1-methyladenine,2-methyladenine, 7-methyladenine, N6-methyladenine,N6,N6-dimethyladenine, 1-methylguanine, 7-methylguanine,N2-methylguanine, and N2,N2-dimethylguanine.

The term “primer,” as used herein, generally refers to a nucleic acidmolecule (e.g., an oligonucleotide), which can include a sequencecomprising A, C, G, T and/or U, or variants or analogs thereof. A primercan be a synthetic oligonucleotide comprising DNA, RNA, PNA, or variantsor analogs thereof. A primer can be designed such that its nucleotidesequence is complementary to a target strand, or the primer can comprisea random nucleotide sequence. In some embodiments, a primer can comprisea tail (e.g., a poly-A tail, an index adaptor, a molecular barcode,etc.). In some embodiments, a primer can comprise 5 to 15 bases, 10 to20 bases, 15 to 25 bases, 20 to 30 bases, 25 to 35 bases, 30 to 40bases, 35 to 45 bases, 40 to 50 bases, 45 to 55 bases, 50 to 60 bases,55 to 65 bases, 60 to 70 bases, 65 to 75 bases, 70 to 80 bases, 75 to 85bases, 80 to 90 bases, 85 to 95 bases, 90 to 100 bases, 95 to 105 bases,100 to 150 bases, 125 to 175 bases, 150 to 200 bases, or more than 200bases.

Luminescent Properties

As described herein, a luminescent molecule is a molecule that absorbsone or more photons and may subsequently emit one or more photons afterone or more time durations. The luminescence of the molecule isdescribed by several parameters, including but not limited toluminescent lifetime, absorption spectra, emission spectra, luminescentquantum yield, and luminescent intensity. The terms absorption andexcitation are used interchangeably throughout the application. Atypical luminescent molecule may absorb, or undergo excitation by, lightat multiple wavelengths. Excitation at certain wavelengths or withincertain spectral ranges may relax by a luminescent emission event, whileexcitation at certain other wavelengths or spectral ranges may not relaxby a luminescent emission event. In some embodiments, a luminescentmolecule is only suitably excited for luminescence at a singlewavelength or within a single spectral range. In some embodiments, aluminescent molecule is suitably excited for luminescence at two or morewavelengths or within two or more spectral ranges. In some embodiments,a molecule is identified by measuring the wavelength of the excitationphoton or the absorption spectrum.

The emitted photon from a luminescent emission event will emit at awavelength within a spectral range of possible wavelengths. Typicallythe emitted photon has a longer wavelength (e.g., has less energy or isred-shifted) compared to the wavelength of the excitation photon. Incertain embodiments, a molecule is identified by measuring thewavelength of an emitted photon. In certain embodiments, a molecule isidentified by measuring the wavelength of a plurality of emitted photon.In certain embodiments, a molecule is identified by measuring theemission spectrum.

Luminescent lifetime refers to the time duration between an excitationevent and an emission event. In some embodiments, luminescent lifetimeis expressed as the constant in an equation of exponential decay. Insome embodiments, wherein there are one or more pulse events deliveringexcitation energy, the time duration is the time between the pulse andthe subsequent emission event.

“Determining a luminescent lifetime” of a molecule can be performedusing any suitable method (e.g., by measuring the lifetime using asuitable technique or by determining time-dependent characteristics ofemission). In some embodiments, determining the luminescent lifetime ofa molecule comprises determining the lifetime relative to one or moremolecules (e.g., different luminescently labeled nucleotides in asequencing reaction). In some embodiments, determining the luminescentlifetime of a molecule comprises determining the lifetime relative to areference. In some embodiments, determining the luminescent lifetime ofa molecule comprises measuring the lifetime (e.g., fluorescencelifetime). In some embodiments, determining the luminescent lifetime ofa molecule comprises determining one or more temporal characteristicsthat are indicative of lifetime. In some embodiments, the luminescentlifetime of a molecule can be determined based on a distribution of aplurality of emission events (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, ormore emission events) occurring across one or more time-gated windowsrelative to an excitation pulse. For example, a luminescent lifetime ofa single molecule can be distinguished from a plurality of moleculeshaving different luminescent lifetimes based on the distribution ofphoton arrival times measured with respect to an excitation pulse.

It should be appreciated that a luminescent lifetime of a singlemolecule is indicative of the timing of photons emitted after the singlemolecule reaches an excited state and the single molecule can bedistinguished by information indicative of the timing of the photons.Some embodiments may include distinguishing a molecule from a pluralityof molecules based on the molecule's luminescent lifetime by measuringtimes associated with photons emitted by the molecule. The distributionof times may provide an indication of the luminescent lifetime which maybe determined from the distribution. In some embodiments, the singlemolecule is distinguishable from the plurality of molecules based on thedistribution of times, such as by comparing the distribution of times toa reference distribution corresponding to a known molecule. In someembodiments, a value for the luminescent lifetime is determined from thedistribution of times.

Luminescent quantum yield refers to the fraction of excitation events ata given wavelength or within a given spectral range that lead to anemission event, and is typically less than 1. In some embodiments, theluminescent quantum yield of a molecule described herein is between 0and about 0.001, between about 0.001 and about 0.01, between about 0.01and about 0.1, between about 0.1 and about 0.5, between about 0.5 and0.9, or between about 0.9 and 1. In some embodiments, a molecule isidentified by determining or estimating the luminescent quantum yield.

As used herein for single molecules, luminescent intensity refers to thenumber of emitted photons per unit time that are emitted by a moleculewhich is being excited by delivery of a pulsed excitation energy. Insome embodiments, the luminescent intensity refers to the detectednumber of emitted photons per unit time that are emitted by a moleculewhich is being excited by delivery of a pulsed excitation energy, andare detected by a particular sensor or set of sensors.

The luminescent lifetime, luminescent quantum yield, and luminescentintensity may each vary for a given molecule under different conditions.In some embodiments, a single molecule will have a different observedluminescent lifetime, luminescent quantum yield, or luminescentintensity than for an ensemble of the molecules. In some embodiments, amolecule confined in a sample well (e.g., a nanoaperture) will have adifferent observed luminescent lifetime, luminescent quantum yield, orluminescent intensity than for molecules not confined in a sample well.In some embodiments, a luminescent label or luminescent moleculeattached to another molecule will have a different luminescent lifetime,luminescent quantum yield, or luminescent intensity than the luminescentlabel or luminescent molecule not attached to another molecule. In someembodiments, a molecule interacting with a macromolecular complex (e.g.,protein complex (e.g., nucleic acid polymerase)) will have differentluminescent lifetime, luminescent quantum yield, or luminescentintensity than a molecule not interacting with a macromolecular complex.

In certain embodiments, a luminescent molecule described in theapplication absorbs one photon and emits one photon after a timeduration. In some embodiments, the luminescent lifetime of a moleculecan be determined or estimated by measuring the time duration. In someembodiments, the luminescent lifetime of a molecule can be determined orestimated by measuring a plurality of time durations for multiple pulseevents and emission events. In some embodiments, the luminescentlifetime of a molecule can be differentiated amongst the luminescentlifetimes of a plurality of types of molecules by measuring the timeduration. In some embodiments, the luminescent lifetime of a moleculecan be differentiated amongst the luminescent lifetimes of a pluralityof types of molecules by measuring a plurality of time durations formultiple pulse events and emission events. In certain embodiments, amolecule is identified or differentiated amongst a plurality of types ofmolecules by determining or estimating the luminescent lifetime of themolecule. In certain embodiments, a molecule is identified ordifferentiated amongst a plurality of types of molecules bydifferentiating the luminescent lifetime of the molecule amongst aplurality of the luminescent lifetimes of a plurality of types ofmolecules.

In certain embodiments, the luminescent emission event is afluorescence. In certain embodiments, the luminescent emission event isa phosphorescence. As used herein, the term luminescence encompasses allluminescent events including both fluorescence and phosphorescence.

In one aspect, the application provides a method of determining theluminescent lifetime of a single luminescent molecule comprising:providing the luminescent molecule in a target volume; delivering aplurality of pulses of an excitation energy to a vicinity of the targetvolume; and detecting a plurality of luminescences from the luminescentmolecule. In some embodiments, the method further comprises evaluatingthe distribution of the plurality of time durations between each pair ofpulses and luminescences. In some embodiments, the method furthercomprises immobilizing the single luminescent molecule in the targetvolume.

In another aspect, the application provides a method of determining theluminescent lifetime of a plurality of molecules comprising: providing aplurality of luminescent molecules in a target volume; delivering aplurality of pulses of an excitation energy to a vicinity of the targetvolume; and detecting a plurality of luminescences from the luminescentmolecules. In some embodiments, the method further comprises evaluatingthe distribution of the plurality of time durations between each pair ofpulses and luminescences. In some embodiments, the method furthercomprises immobilizing the luminescent molecules in the target volume.In some embodiments, the plurality consists of between 2 and about 10molecules, between about 10 and about 100 molecules, or between about100 and about 1000 molecules. In some embodiments, the pluralityconsists of between about 1000 and about 10⁶ molecules, between about10⁶ and about 10⁹ molecules, between about 10⁹ and about 10¹² molecules,between about 10¹² and about 10¹⁵ molecules, or between about 10¹⁵ andabout 10¹⁸ molecules. In some embodiments, all molecules of theplurality are the same type of molecule.

FIG. 3 shows the exemplary decay profile 3-1 for four luminescentmolecules with different luminescent lifetimes (longest to shortest, topto bottom). The amplitude can refer to the intensity of luminescencefrom a sample comprising many molecules, which decreases exponentiallyover time after the initial excitation based on the luminescentlifetime. The amplitude can alternatively refer to a number or count ofemissions detected after a time duration after a plurality of pulses ofexcitation energy, for example, for a single molecule. The normalizedcumulative distribution function 3-2 corresponds to 3-1, for fourluminescent molecules with different luminescent lifetimes (shortest tolongest, top to bottom). The CDF can represent the normalizedprobability of the luminescence amplitude of reaching zero (e.g., thecumulative probability of all excited molecules having luminesced) overtime after the initial excitation based on the luminescent lifetime. TheCDF can alternatively represent the normalized probability of a singlemolecule emitting luminescence at a certain time duration after a singlepulse or after each of a plurality of pulses of excitation energy.

In one aspect, the application provides a method of determining theluminescent intensity of a single luminescent molecule comprising:providing the luminescent molecule in a target volume; delivering aplurality of pulses of an excitation energy to a vicinity of the targetvolume; and detecting a plurality of luminescences from the luminescentmolecule. In some embodiments, the method further comprises determiningthe number of the plurality of detected luminescence per unit time. Insome embodiments, the method further comprises immobilizing the singleluminescent molecule in the target volume.

In another aspect, the application provides a method of determining theluminescent intensity of a plurality of molecules comprising: providinga plurality of luminescent molecules in a target volume; delivering aplurality of pulses of an excitation energy to a vicinity of the targetvolume; and detecting a plurality of luminescences from the luminescentmolecules. In some embodiments, the method further comprises determiningthe number of the plurality of detected luminescence per unit time. Insome embodiments, the method further comprises immobilizing theluminescent molecules in the target volume. In some embodiments, theplurality consists of between 2 and about 10 molecules, between about 10and about 100 molecules, or between about 100 and about 1000 molecules.In some embodiments, the plurality consists of between about 1000 andabout 10⁶ molecules, between about 10⁶ and about 10⁹ molecules, betweenabout 10⁹ and about 10¹² molecules, between about 10¹² and about 10¹⁵molecules, or between about 10¹⁵ and about 10¹⁸ molecules. In someembodiments, all molecules of the plurality are the same type ofmolecule.

Excitation Energy

In one aspect of methods described herein, one or more excitation energyis used to excite the luminescent labels of the molecules to beidentified or distinguished. In some embodiments, an excitation energyis in the visible spectrum. In some embodiments, an excitation energy isin the ultraviolet spectrum. In some embodiments, an excitation energyis in the infrared spectrum. In some embodiments, one excitation energyis used to excite the luminescently labeled molecules. In someembodiments, two excitation energies are used to excite theluminescently labeled molecules. In some embodiments, three or moreexcitation energies are used to excite the luminescently labeledmolecules. In some embodiments, each luminescently labeled molecule isexcited by only one of the delivered excitation energies. In someembodiments, a luminescently labeled molecule is excited by two or moreof the delivered excitation energies. In certain embodiments, anexcitation energy may be monochromatic or confined to a spectral range.In some embodiments, a spectral range has a range of between about 0.1nm and about 1 nm, between about 1 nm and about 2 nm, or between about 2nm and about 5 nm. In some embodiments a spectral range has a range ofbetween about 5 nm and about 10 nm, between about 10 nm and about 50 nm,or between about 50 nm and about 100 nm.

In certain embodiments, excitation energy is delivered as a pulse oflight. In certain embodiments, excitation energy is delivered as aplurality of pulses of light. In certain embodiments, two or moreexcitation energies are used to excite the luminescently labeledmolecules. In some embodiments, each excitation energy is delivered atthe same time (e.g., in each pulse). In some embodiments, eachexcitation energy is delivered at different times (e.g., in separatepulses of each energy). The different excitation energies may bedelivered in any pattern sufficient to allow detection of luminescencefrom the target molecules. In some embodiments, two excitation energiesare delivered in each pulse. In some embodiments, a first excitationenergy and a second excitation energy are delivered in alternatingpulses. In some embodiments, a first excitation energy is delivered in aseries of sequential pulses, and a second excitation energy is deliveredin a subsequent series of sequential pulses, or an alternating patternof such series.

In certain embodiments, the frequency of pulses of light is selectedbased on the luminescent properties of the luminescently labeledmolecule. In certain embodiments, the frequency of pulses of light isselected based on the luminescent properties of a plurality ofluminescently labeled nucleotides. In certain embodiments, the frequencyof pulses of light is selected based on the luminescent lifetime of aplurality of luminescently labeled nucleotides. In some embodiments, thefrequency is selected so that the gap between pulses is longer than theluminescent lifetimes of one or more luminescently labeled nucleotides.In some embodiments, the frequency is selected based on the longestluminescent lifetime of the plurality of luminescently labelednucleotides. For example, if the luminescent lifetimes of the fourluminescently labeled nucleotides are 0.25, 0.5, 1.0, and 1.5 ns, thefrequency of pulses of light may be selected so that the gap betweenpulses exceeds 1.5 ns. In some embodiments, the gap is between about twotimes and about ten times, between about ten times and about 100 times,or between about 100 times and about 1000 times longer than theluminescent lifetime of one or more luminescently labeled moleculesbeing excited. In some embodiments, the gap is about 10 times longerthan the luminescent lifetime of one or more luminescently labeledmolecules being excited. In some embodiments, the gap is between about0.01 ns and about 0.1 ns, between about 1 ns and about 5 ns, betweenabout 5 ns and about 15 ns, between about 15 ns and about 25 ns, orbetween about 25 ns and about 50 ns. In some embodiments, the gap isselected such that there is a 50%, 75%, 90%, 95%, or 99% probabilitythat the molecules excited by the pulse will luminescently decay or thatthe excited state will relax by another mechanism.

In certain embodiments, wherein there are multiple excitation energies,the frequency of the pulses for each excitation energy is the same. Incertain embodiments, wherein there are multiple excitation energies, thefrequencies of the pulses for each excitation energy is different. Forexample, if a red laser is used to excite luminescent molecules withlifetimes of 0.2 and 0.5 ns, and a green laser is used to exciteluminescent molecules with lifetimes of 5 ns and 7 ns, the gap aftereach red laser pulse may be shorter (e.g., 5 ns) than the gap after eachgreen laser pulse (e.g., 20 ns).

In certain embodiments, the frequency of pulsed excitation energies isselected based on the chemical process being monitored. For a sequencingreaction the frequency may be selected such that a number of pulses aredelivered sufficient to allow for detection of a sufficient number ofemitted photons to be detected. A sufficient number, in the context ofdetected photons, refers to a number of photons necessary to identify ordistinguish the luminescently labeled nucleotide from the plurality ofluminescently labeled nucleotides. For example, a DNA polymerase mayincorporate an additional nucleotide once every 20 milliseconds onaverage. The time that a luminescently labeled nucleotide interacts withthe complex may be about 10 milliseconds, and the time between when theluminescent marker is cleaved and the next luminescently labelednucleotide begins to interact may be about 10 milliseconds. Thefrequency of the pulsed excitation energy could then be selected todeliver sufficient pulses over 10 milliseconds such that a sufficientnumber of emitted photons are detected during the 10 millisecond whenthe luminescently labeled nucleotide is being incorporated. For example,at a frequency of 100 MHz, there will be 1 million pulses in 10milliseconds (the approximate length of the incorporation event). If0.1% of these pulses leads to a detected photon there will be 1,000luminescent data points that can be analyzed to determine the identityof the luminescently labeled nucleotide being incorporated. Any of theabove values are non-limiting. In some embodiments incorporation eventsmay take between 1 ms and 20 ms, between 20 ms and 100 ms, or between100 ms and 500 ms. In some embodiments, in which multiple excitationenergies are delivered in separately timed pulses the luminescentlylabeled nucleotide may only be excited by a portion of the pulses. Insome embodiments, the frequency and pattern of the pulses of multipleexcitation energies is selected such that the number of pulses issufficient to excite any one of the plurality of luminescently labelednucleotides to allow for a sufficient number of emitted photons to bedetected.

In some embodiments, the frequency of pulses is between about 1 MHz andabout 10 MHz. In some embodiments, the frequency of pulses is betweenabout 10 MHz and about 100 MHz. In some embodiments, the frequency ofpulses is between about 100 MHz and about 1 GHz. In some embodiments,the frequency of pulses is between about 50 MHz and about 200 MHz. Insome embodiments, the frequency of pulses is about 100 MHz. In someembodiments, the frequency is stochastic.

In certain embodiments, the excitation energy is between about 500 nmand about 700 nm. In some embodiments, the excitation energy is betweenabout 500 nm and about 600 nm, or about 600 nm and about 700 nm. In someembodiments, the excitation energy is between about 500 nm and about 550nm, between about 550 nm and about 600 nm, between about 600 nm andabout 650 nm, or between about 650 nm and about 700 nm.

In certain embodiments, a method described herein comprises delivery oftwo excitation energies. In some embodiments, the two excitationenergies are separated by between about 5 nm and about 20 nm, betweenabout 20 nm and about 40 nm, between about 40 nm and about 60 nm,between about 60 nm and about 80 nm, between about 80 nm and about 100nm, between about 100 nm and about 150 nm, between about 150 nm andabout 200 nm, between about 200 nm and about 400 nm, or between at leastabout 400 nm. In some embodiments, the two excitation energies areseparated by between about 20 nm and about 80 nm, or between about 80 nmand about 160 nm.

When an excitation energy is referred to as being in a specific range,the excitation energy may comprise a single wavelength, such that thewavelength is between or at the endpoints of the range, or theexcitation energy may comprise a spectrum of wavelengths with a maximumintensity, such that the maximum intensity is between or at theendpoints of the range.

In certain embodiments, the first excitation energy is in the range of450 nm to 500 nm and the second excitation energy is in the range of 500nm to 550 nm, 550 nm to 600 nm, 600 nm to 650 nm, or 650 nm to 700 nm.In certain embodiments, the first excitation energy is in the range of500 nm to 550 nm and the second excitation energy is in the range of 450nm to 500 nm, 550 nm to 600 nm, 600 nm to 650 nm, or 650 nm to 700 nm.In certain embodiments, the first excitation energy is in the range of550 nm to 600 nm and the second excitation energy is in the range of 450nm to 500 nm, 500 nm to 550 nm, 600 nm to 650 nm, or 650 nm to 700 nm.In certain embodiments, the first excitation energy is in the range of600 nm to 650 nm and the second excitation energy is in the range of 450nm to 500 nm, 500 nm to 550 nm, 550 nm to 600 nm, or 650 nm to 700 nm.In certain embodiments, the first excitation energy is in the range of650 nm to 700 nm and the second excitation energy is in the range of 450nm to 500 nm, 500 nm to 550 nm, 550 nm to 600 nm, or 600 nm to 650 nm.

In certain embodiments, the first excitation energy is in the range of450 nm to 500 nm and the second excitation energy is in the range of 500nm to 550 nm. In certain embodiments, the first excitation energy is inthe range of 450 nm to 500 nm and the second excitation energy is in therange of 550 nm to 600 nm. In certain embodiments, the first excitationenergy is in the range of 450 nm to 500 nm and the second excitationenergy is in the range of 600 nm to 670 nm. In certain embodiments, thefirst excitation energy is in the range of 500 nm to 550 nm and thesecond excitation energy is in the range of 550 nm to 600 nm. In certainembodiments, the first excitation energy is in the range of 500 nm to550 nm and the second excitation energy is in the range of 600 nm to 670nm. In certain embodiments, the first excitation energy is in the rangeof 550 nm to 600 nm and the second excitation energy is in the range of600 nm to 670 nm. In certain embodiments, the first excitation energy isin the range of 470 nm to 510 nm and the second excitation energy is inthe range of 510 nm to 550 nm. In certain embodiments, the firstexcitation energy is in the range of 470 nm to 510 nm and the secondexcitation energy is in the range of 550 nm to 580 nm. In certainembodiments, the first excitation energy is in the range of 470 nm to510 nm and the second excitation energy is in the range of 580 nm to 620nm. In certain embodiments, the first excitation energy is in the rangeof 470 nm to 510 nm and the second excitation energy is in the range of620 nm to 670 nm. In certain embodiments, the first excitation energy isin the range of 510 nm to 550 nm and the second excitation energy is inthe range of 550 nm to 580 nm. In certain embodiments, the firstexcitation energy is in the range of 510 nm to 550 nm and the secondexcitation energy is in the range of 580 nm to 620 nm. In certainembodiments, the first excitation energy is in the range of 510 nm to550 nm and the second excitation energy is in the range of 620 nm to 670nm. In certain embodiments, the first excitation energy is in the rangeof 550 nm to 580 nm and the second excitation energy is in the range of580 nm to 620 nm. In certain embodiments, the first excitation energy isin the range of 550 nm to 580 nm and the second excitation energy is inthe range of 620 nm to 670 nm. In certain embodiments, the firstexcitation energy is in the range of 580 nm to 620 nm and the secondexcitation energy is in the range of 620 nm to 670 nm.

Certain embodiments of excitation energy sources and devices fordelivery of excitation energy pulses to a target volume are describedelsewhere herein.

Luminescently Labeled Nucleotides

In one aspect, methods and compositions described herein comprises oneor more luminescently labeled nucleotides. In certain embodiments, oneor more nucleotides comprise deoxyribose nucleosides. In someembodiments, all nucleotides comprises deoxyribose nucleosides. Incertain embodiments, one or more nucleotides comprise ribosenucleosides. In some embodiments, all nucleotides comprise ribosenucleosides. In some embodiments, one or more nucleotides comprise amodified ribose sugar or ribose analog (e.g., a locked nucleic acid). Insome embodiments, one or more nucleotides comprise naturally occurringbases (e.g., cytosine, guanine, adenine, thymine, uracil). In someembodiments, one or more nucleotides comprise derivatives or analogs ofcytosine, guanine, adenine, thymine, or uracil.

In certain embodiments, a method comprises the step of exposing apolymerase complex to a plurality of luminescently labeled nucleotides.In certain embodiments, a composition or device comprises a reactionmixture comprising a plurality of luminescently labeled nucleotides. Insome embodiments, the plurality of nucleotides comprises four types ofnucleotides. In some embodiments, the four types of nucleotides eachcomprise one of cytosine, guanine, adenine, and thymine. In someembodiments, the four types of nucleotides each comprise one ofcytosine, guanine, adenine, and uracil.

In certain embodiments, the concentration of each type of luminescentlylabeled nucleotide in the reaction mixture is between about 50 nM andabout 200 nM, about 200 nM and about 500 nM, about 500 nM and about 1μM, about 1 μM and about 50 μM, or about 50 μM and 250 μM. In someembodiments, the concentration of each type of luminescently labelednucleotide in the reaction mixture is between about 250 nM and about 2μM. In some embodiments, the concentration of each type of luminescentlylabeled nucleotide in the reaction mixture is about 1 μM.

In certain embodiments, the reaction mixture contains additionalreagents of use for sequencing reactions. In some embodiments, thereaction mixture comprises a buffer. In some embodiments, a buffercomprises 3-(N-morpholino)propanesulfonic acid (MOPS). In someembodiments, a buffer is present in a concentration of between about 1mM and between about 100 mM. In some embodiments, the concentration ofMOPS is about 50 mM. In some embodiments, the reaction mixture comprisesone or more salt. In some embodiments, a salt comprises potassiumacetate. In some embodiments, the concentration of potassium acetate isabout 140 mM. In some embodiments, a salt is present in a concentrationof between about 1 mM and about 200 mM. In some embodiments, thereaction mixture comprises a magnesium salt (e.g., magnesium acetate).In some embodiments, the concentration of magnesium acetate is about 20mM. In some embodiments, a magnesium salt is present in a concentrationof between about 1 mM and about 50 mM. In some embodiments, the reactionmixture comprises a reducing agent. In some embodiments, a reducingagent is dithiothreitol (DTT). In some embodiments, a reducing agent ispresent in a concentration of between about 1 mM and about 50 mM. Insome embodiments, the concentration of DTT is about 5 mM. In someembodiments, the reaction mixture comprises one or photostabilizers. Insome embodiments, the reaction mixture comprises an anti-oxidant, oxygenscavenger, or triplet state quencher. In some embodiments, aphotostabilizer comprises protocatechuic acid (PCA). In someembodiments, a photostabilizer comprises 4-nitrobenzyl alcohol (NBA). Insome embodiments, a photostabilizer is present in a concentration ofbetween about 0.1 mM and about 20 mM. In some embodiments, theconcentration of PCA is about 3 mM. In some embodiments, theconcentration of NBA is about 3 mM. A mixture with a photostabilizer(e.g., PCA) may also comprise an enzyme to regenerate thephotostabilizer (e.g., protocatechuic acid dioxygenase (PCD)). In someembodiments, the concentration of PCD is about 0.3 mM.

The application contemplates different methods for differentiatingnucleotides amongst a plurality of nucleotides. In certain embodiments,each of the luminescently labeled nucleotides has a differentluminescent lifetime. In certain embodiments, two or more of theluminescently labeled nucleotides have the same luminescent lifetimes orsubstantially the same luminescent lifetimes (e.g., lifetimes thatcannot be distinguished by the method or device).

In certain embodiments, each of the luminescently labeled nucleotidesabsorbs excitation energy in a different spectral range. In certainembodiments, two of the luminescently labeled nucleotides absorbexcitation energy in the same spectral range. In certain embodiments,three of the luminescently labeled nucleotides absorb excitation energyin the same spectral range. In certain embodiments, four or more of theluminescently labeled nucleotides absorb excitation energy in the samespectral range. In certain embodiments, two of the luminescently labelednucleotides absorb excitation energy a different spectral range. Incertain embodiments, three of the luminescently labeled nucleotidesabsorb excitation energy a different spectral range. In certainembodiments, four or more of the luminescently labeled nucleotidesabsorb excitation energy a different spectral range.

In certain embodiments, each of the luminescently labeled nucleotidesemits photons in a different spectral range. In certain embodiments, twoof the luminescently labeled nucleotides emits photons in the samespectral range. In certain embodiments, three of the luminescentlylabeled nucleotides emits photons in the same spectral range. In certainembodiments, four or more of the luminescently labeled nucleotides emitsphotons in the same spectral range. In certain embodiments, two of theluminescently labeled nucleotides emits photons in the differentspectral range. In certain embodiments, three of the luminescentlylabeled nucleotides emits photons in the different spectral range. Incertain embodiments, four or more of the luminescently labelednucleotides emits photons in the different spectral range.

In certain embodiments, each of four luminescently labeled nucleotideshas a different luminescent lifetime. In certain embodiments, two ormore luminescently labeled nucleotides have different luminescentlifetimes and absorb and/or emit photons in a first spectral range, andone or more luminescently labeled nucleotides absorb and/or emit photonsin a second spectral range. In some embodiments, each of threeluminescently labeled nucleotides has a different luminescent lifetimeand emit luminescence in a first spectral range, and a fourthluminescently labeled nucleotide absorbs and/or emits photons in asecond spectral range. In some embodiments, each of two luminescentlylabeled nucleotides has a different luminescent lifetime and emitluminescence in a first spectral range, and a third and fourthluminescently labeled nucleotide each have different luminescentlifetimes and emit luminescence in a second spectral range.

In certain embodiments, each of four luminescently labeled nucleotideshas a different luminescent intensity. In certain embodiments, two ormore luminescently labeled nucleotides have different luminescentintensity and emit luminescence in a first spectral range, and one ormore luminescently labeled nucleotides absorbs and/or emits photons in asecond spectral range. In some embodiments, each of three luminescentlylabeled nucleotides has a different luminescent intensity and emitluminescence in a first spectral range, and a fourth luminescentlylabeled nucleotide absorbs and/or emits photons in a second spectralrange. In some embodiments, each of two luminescently labelednucleotides has a different luminescent intensity and emit luminescencein a first spectral range, and a third and fourth luminescently labelednucleotide each have different luminescent intensity and emitluminescence in a second spectral range.

In certain embodiments, each of four luminescently labeled nucleotideshas a different luminescent lifetime or luminescent intensity. Incertain embodiments, two or more luminescently labeled nucleotides havedifferent luminescent lifetime or luminescent intensity and emitluminescence in a first spectral range, and one or more luminescentlylabeled nucleotides absorbs and/or emits photons in a second spectralrange. In some embodiments, each of three luminescently labelednucleotides has a different luminescent lifetime or luminescentintensity and emit luminescence in a first spectral range, and a fourthluminescently labeled nucleotide absorbs and/or emits photons in asecond spectral range. In some embodiments, each of two luminescentlylabeled nucleotides has a different luminescent lifetime or luminescentintensity and emit luminescence in a first spectral range, and a thirdand fourth luminescently labeled nucleotide each have differentluminescent lifetime or luminescent intensity and emit luminescence in asecond spectral range.

In certain embodiments, two or more luminescently labeled nucleotideshave different luminescent lifetimes and absorb excitation energy in afirst spectral range, and one or more luminescently labeled nucleotidesabsorbs excitation energy in a second spectral range. In someembodiments, each of three luminescently labeled nucleotides has adifferent luminescent lifetime and absorb excitation energy in a firstspectral range, and a fourth luminescently labeled nucleotide absorbsexcitation energy in a second spectral range. In some embodiments, eachof two luminescently labeled nucleotides has a different luminescentlifetime and absorb excitation energy in a first spectral range, and athird and fourth luminescently labeled nucleotide each have differentluminescent lifetimes and absorb excitation energy in a second spectralrange.

In certain embodiments, two or more luminescently labeled nucleotideshave different luminescent lifetime or luminescent intensity and absorbexcitation energy in a first spectral range, and one or moreluminescently labeled nucleotides absorbs excitation energy in a secondspectral range. In some embodiments, each of three luminescently labelednucleotides has a different luminescent lifetime or luminescentintensity and absorb excitation energy in a first spectral range, and afourth luminescently labeled nucleotide absorbs excitation energy in asecond spectral range. In some embodiments, each of two luminescentlylabeled nucleotides has a different luminescent lifetime or luminescentintensity and absorb excitation energy in a first spectral range, and athird and fourth luminescently labeled nucleotide each have differentluminescent lifetime or luminescent intensity and absorb excitationenergy in a second spectral range.

During sequencing the method of identifying a nucleotide may varybetween various base pairs in the sequence. In certain embodiments, twotypes of nucleotides may be labeled to absorb at a first excitationenergy, and those two types of nucleotides (e.g., A, G) aredistinguished based on different luminescent intensity, whereas twoadditional types of nucleotides (e.g., C, T) may be labeled to absorb ata second excitation energy, and those two additional types ofnucleotides are distinguished based on different luminescent lifetime.For such an embodiment, during sequencing certain segments of thesequence may be determined only based on luminescent intensity (e.g.,segments incorporating only A and G), whereas other segments of thesequence may be determined only based on luminescent lifetime (e.g.,segments incorporating only C and T). In some embodiments, between 2 and4 luminescently labeled nucleotide are be differentiated based onluminescent lifetime. In some embodiments, between 2 and 4 luminescentlylabeled nucleotides are differentiated based on luminescent intensity.In some embodiments, between 2 and 4 luminescently labeled nucleotidesare differentiated based on luminescent lifetime and luminescentintensity.

FIG. 4 shows the luminescent lifetime 4-1 of exemplary luminescentlylabeled nucleotides and the luminescent intensity 4-2 for the sameexemplary nucleotides. For example, the fourth row shows data for adeoxythymidine hexaphosphate (dT6P) nucleotide linked to the fluorophoreAlexa Fluor® 555 (AF555). This luminescently labeled nucleotide has alifetime of approximately 0.25 ns and displays a luminescent intensityof approximately 20000 counts/s. The observed luminescent lifetime andluminescent intensity of any luminescently labeled nucleotide may, ingeneral, differ for the nucleotide under incorporation conditions (e.g.,in a single molecule complex, in a nanoaperture) versus other moretypical conditions such as those for 4-1 and 4-2.

Luminescence Detection

In one aspect of methods described herein, an emitted photon (aluminescence) or a plurality of emitted photons is detected by one ormore sensors. For a plurality of luminescently labeled molecules ornucleotides, each of the molecules may emit photons in a single spectralrange, or a portion of the molecules may emit photons in a firstspectral range and another portion of molecules may emit photons in asecond spectral range. In certain embodiments, the emitted photons aredetected by a single sensor. In certain embodiments, the emitted photonsare detected by multiple sensors. In some embodiments, the photonsemitted in a first spectral range are detected by a first sensor, andthe photons emitted in a second spectral range are detected by a secondsensor. In some embodiments, the photons emitted in each of a pluralityof spectral ranges are detected by a different sensor.

In certain embodiments, each sensor is configured to assign a time binto an emitted photon based on the time duration between the excitationenergy and the emitted photon. In some embodiments, photons emittedafter a shorter time duration will be assigned an earlier time bin, andphotons emitted after a longer duration will be assigned a later timebin.

In some embodiments, a plurality of pulses of excitation energy isdelivered to vicinity of a target volume and a plurality of photons,which may include photon emission events, are detected. In someembodiments, the plurality of luminescences (e.g., photon emissionevents) correspond to incorporation of a luminescently labelednucleotide into a nucleic acid product. In some embodiments, theincorporation of a luminescently labeled nucleotide lasts for betweenabout 1 ms and about 5 ms, between about 5 ms and about 20 ms, betweenabout 20 ms and about 100 ms, or between about 100 ms and about 500 ms.In some embodiments, between about 10 and about 100, between about 100and about 1000, about 1000 and about 10000, or about 10000 and about100000 luminescences are detected during incorporation of aluminescently labeled nucleotide.

In certain embodiments, there are no luminescences detected if aluminescently labeled nucleotide is not being incorporated. In someembodiments, there is a luminescence background. In some embodiments,spurious luminescences are detected when no luminescently labelednucleotide is being incorporated. Such spurious luminescences may occurif one or more luminescently labeled nucleotides is in the target volume(e.g., diffuses into the target volume, or interacts with polymerase butis not incorporated) during a pulse of excitation energy, but is notbeing incorporated by the sequencing reaction. In some embodiments, theplurality of luminescences detected from a luminescently labelednucleotide in the target volume but not being incorporated is smaller(e.g., ten times, 100 times, 1000 times, 10000 times) than the pluralityof luminescences from a luminescently labeled nucleotide.

In some embodiments, for each plurality of detected luminescencescorresponding to incorporation of a luminescently labeled nucleotide theluminescences are assigned a time bin based on the time duration betweenthe pulse and the emitted photon. This plurality for an incorporationevent is referred to herein as a “burst”. In some embodiments, a burstrefers to a series of signals (e.g., measurements) above a baseline(e.g., noise threshold value), wherein the signals correspond to aplurality of emission events that occur when the luminescently labelednucleotide is within the excitation region. In some embodiments, a burstis separated from a preceding and/or subsequent burst by a time intervalof signals representative of the baseline. In some embodiments, theburst is analyzed by determining the luminescent lifetime based on theplurality of time durations. In some embodiments, the burst is analyzedby determining the luminescent intensity based on the number of detectedluminescences per a unit of time. In some embodiments, the burst isanalyzed by determining the spectral range of the detectedluminescences. In some embodiments, analyzing the burst data will allowassignment of the identity of the incorporated luminescently labelednucleotide, or allow one or more luminescently labeled nucleotides to bedifferentiated from amongst a plurality of luminescently labelednucleotides. The assignment or differentiation may rely on any one ofluminescent lifetime, luminescent intensity, spectral range of theemitted photons, or any combination thereof.

FIG. 5 depicts the sequencing of an exemplary template nucleic acid. Thesequencing experiment was run with 4 luminescently labeled nucleotides:deoxyadeno sine linked to Alexa Fluor® 647 (A-AF647), dexoythymidinelinked to Alex Fluor 555 (T-AF555), deoxyguanidine linked to DyLight®554-R1 (G-D554R1), and dexoycytidine linked to DyLight® 530-R2(C-D530R2). The nucleotide A-AF647 is excited by excitation energy inthe red spectral range, and T, G, and C nucleotides are excited byexcitation in the green spectral range. The number of photons detectedover ˜200 s of a sequencing reaction are shown in an intensity trace5-1. Each spike corresponds to a burst of detected luminescences and ismarked with a dot. Each burst may correspond to the incorporation of aluminescently labeled nucleotide, and comprises thousands of detectedluminescences. Different colored traces can be utilized to denotedifferent excitation pulses. For example, a purple trace can be used forgreen excitation pulses, and a blue trace can be used for red excitationpulses. Bursts from the blue trace can be assigned to the incorporationof the nucleotide A-AF647 (the only nucleotide with red luminescentmolecule in this example).

FIG. 5 shows one way of reducing the raw data to differentiate bursts ofthe same color (e.g., bursts in the purple trace between T, G, and C)using an intensity versus lifetime plot 5-2. Each circle represents aburst from the purple trace. Each burst has been analyzed to determinethe luminescent lifetime of the luminescently labeled nucleotide basedon the time duration between pulse and emission of each detected photon.Additionally, each burst has been analyzed to determine the luminescentintensity of the luminescently labeled nucleotide based on the number ofdetected photons per second. The incorporation events are clustered inthree groups corresponding to each of the three luminescently labelednucleotides. The dark cluster in the lower portion of the plot (area ofthe plot below the dashed line) is assigned to C-D530R2 which has thelongest luminescent lifetime and the lowest luminescent intensity. Thelight cluster in the lower portion of the plot (area of the plot belowthe dashed line) is assigned to G-D554R1 which has the intermediatelifetime and intensity. And the light cluster in the upper portion ofthe plot (area of the plot above the dashed line) is assigned to T-AF555which has the shortest lifetime and highest intensity. FIG. 5 shows thealignment 5-3 between the sequence determined from the data and theknown sequence of the template nucleic acid. Vertical bars indicate amatch between the experimentally determined base and the targetsequence. Dashes indicate a position in the template sequence for whichno nucleotide was assigned in the determined sequence, or an extraposition in the determined sequence which does not correspond to anyposition in the template sequence.

FIG. 6 depicts a second example for sequencing of a template nucleicacid. The sequencing experiment was run with 4 luminescently labelednucleotides: deoxyadenosine linked to Alexa Fluor® 647 (A-AF647),dexoythymidine linked to Alex Fluor 555 (T-AF555), deoxyguanidine linkedto Alexa Fluor® 647 (G-AF647), and a dexoycytidine linked to AlexaFluor® 546 (C-AF546). The nucleotides A-AF647 and G-AF647 are excited byexcitation energy in the red spectral range, and T and C nucleotides areexcited by excitation in the green spectral range. In this experiment, Aand G have the same luminescent marker, and are not discriminated. FIG.6 shows the number of photons detected over ˜300 s of a sequencingreaction in an intensity trace 6-1. Each spike corresponds to a burst ofdetected luminescences and is marked with a dot. Each burst maycorrespond to the incorporation of a luminescently labeled nucleotide,and comprises thousands of detected luminescences. The trace showsdetected luminescences for green excitation pulses (corresponding tobases T and C).

FIG. 6 shows one way of reducing the raw data to differentiate T and Cusing an intensity versus lifetime plot 6-2. Each circle represents aburst from the intensity trace 6-1. Each burst has been analyzed todetermine the luminescent lifetime of the luminescently labelednucleotide based on the time duration between pulse and emission of eachdetected photon. Additionally each burst has been analyzed to determinethe luminescent intensity of the luminescently labeled nucleotide basedon the number of detected photons per second. The incorporation eventsare clustered in two groups corresponding to each of the twoluminescently labeled nucleotides. The dark cluster in the right portionof the plot (area of the plot to the right of the dashed line) isassigned to C-AF546 which has the longest luminescent lifetime and thelowest luminescent intensity. The light cluster in the right portion ofthe plot (area of the plot to the right of the dashed line) is assignedto T-AF555 which has the shortest lifetime and highest intensity. FIG. 6shows the alignment 6-3 between the sequence determined from the dataand the known sequence of the template nucleic acid. Vertical barsindicate a match between the experimentally determined base and thetarget sequence. Dashes indicate a position in the template sequence forwhich no nucleotide was assigned in the determined sequence, or an extraposition in the determined sequence which does not correspond to anyposition in the template sequence.

In some embodiments, one or more components of a sequencing reaction canbe prepared and used in a sample well, as depicted in the non-limitingembodiments shown in FIGS. 7-9, for example, for the analysis ofluminescent labels in the context of a sequencing reaction.

Luminescent Labels

The terms luminescent tag, luminescent label and luminescent marker areused interchangeably throughout, and relate to molecules comprising oneor more luminescent molecules. In certain embodiments, the incorporatedmolecule is a luminescent molecule, e.g., without attachment of adistinct luminescent label. Typical nucleotide and amino acids are notluminescent, or do not luminesce within suitable ranges of excitationand emission energies. In certain embodiments, the incorporated moleculecomprises a luminescent label. In certain embodiments, the incorporatedmolecule is a luminescently labeled nucleotide. In certain embodiments,the incorporated molecule is a luminescently labeled amino acid orluminescently labeled tRNA. In some embodiments, a luminescently labelednucleotide comprises a nucleotide and a luminescent label. In someembodiments, a luminescently labeled nucleotide comprises a nucleotide,a luminescent label, and a linker. In some embodiments, the luminescentlabel is a fluorophore.

In certain embodiments, the luminescent label, and optionally thelinker, remain attached to the incorporated molecule. In certainembodiments, the luminescent label, and optionally the linker, arecleaved from the molecule during or after the process of incorporation.

In certain embodiments, the luminescent label is a cyanine dye, or ananalog thereof. In some embodiments, the cyanine dye is of formula:

or a salt, stereoisomer, or tautomer thereof, wherein:

-   -   A¹ and A² are joined to form an optionally substituted, aromatic        or non-aromatic, monocyclic or polycyclic, heterocyclic ring;    -   B¹ and B² are joined to form an optionally substituted, aromatic        or non-aromatic, monocyclic or polycyclic, heterocyclic ring;    -   each of R¹ and R² is independently hydrogen, optionally        substituted alkyl; and    -   each of L¹ and L² is independently hydrogen, optionally        substituted alkyl, or L¹ and L² are joined to form an optionally        substituted, aromatic or non-aromatic, monocyclic or polycyclic,        carbocyclic ring.

In certain embodiments, the luminescent label is a rhodamine dye, or ananalog thereof. In some embodiments, the rhodamine dye is of formula:

or a salt, stereoisomer, or tautomer thereof, wherein:

-   -   each of A¹ and A² is independently hydrogen, optionally        substituted alkyl, optionally substituted aromatic or        non-aromatic heterocyclyl, optionally substituted aromatic or        non-aromatic carbocyclyl, or optionally substituted carbonyl, or        A¹ and A² are joined to form an optionally substituted, aromatic        or non-aromatic, monocyclic or polycyclic, heterocyclic ring;    -   each of B¹ and B² is independently hydrogen, optionally        substituted alkyl, optionally substituted, aromatic or        non-aromatic heterocyclyl, optionally substituted, aromatic or        non-aromatic carbocyclyl, or optionally substituted carbonyl, or        B¹ and B² are joined to form an optionally substituted, aromatic        or non-aromatic, monocyclic or polycyclic, heterocyclic ring;    -   each of R² and R³ is independently hydrogen, optionally        substituted alkyl, optionally substituted aryl, or optionally        substituted acyl; and    -   R⁴ is hydrogen, optionally substituted alkyl, optionally        substituted, optionally substituted aromatic or non-aromatic        heterocyclyl, optionally substituted aromatic or non-aromatic        carbocyclyl, or optionally substituted carbonyl.        In some embodiments, R⁴ is optionally substituted phenyl. In        some embodiments, R⁴ is optionally substituted phenyl, wherein        at least one substituent is optionally substituted carbonyl. In        some embodiments, R⁴ is optionally substituted phenyl, wherein        at least one substituent is optionally substituted sulfonyl.

Typically, the luminescent label comprises an aromatic or heteroaromaticcompound and can be a pyrene, anthracene, naphthalene, acridine,stilbene, indole, benzindole, oxazole, carbazole, thiazole,benzothiazole, phenanthridine, phenoxazine, porphyrin, quinoline,ethidium, benzamide, cyanine, carbocyanine, salicylate, anthranilate,coumarin, fluoroscein, rhodamine or other like compound. Exemplary dyesinclude xanthene dyes, such as fluorescein or rhodamine dyes, including5-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE),tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G),N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine(ROX). Exemplary dyes also include naphthylamine dyes that have an aminogroup in the alpha or beta position. For example, naphthylaminocompounds include 1-dimethylaminonaphthyl-5-sulfonate,1-anilino-8-naphthalene sulfonate and 2-p-toluidinyl-6-naphthalenesulfonate, 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Other exemplary dyes include coumarins, such as3-phenyl-7-isocyanatocoumarin; acridines, such as9-isothiocyanatoacridine and acridine orange;N-(p-(2-benzoxazolyl)phenyl)maleimide; cyanines, such asindodicarbocyanine 3 (Cy®3),(2Z)-2-[(E)-3-[3-(5-carboxypentyl)-1,1-dimethyl-6,8-disulfobenzo[e]indol-3-ium-2-yl]prop-2-enylidene]-3-ethyl-1,1-dimethyl-8-(trioxidanylsulfanyl)benzo[e]indole-6-sulfonate(Cy®3.5),2-{2-[(2,5-dioxopyrrolidin-1-yl)oxy]-2-oxoethyl}-16,16,18,18-tetramethyl-6,7,7a,8a,9,10,16,18-octahydrobenzo[2″,3″]indolizino[8″,7″:5′,6′]pyrano[3′,2′: 3,4]pyrido[1,2-a]indol-5-ium-14-sulfonate (Cy®3B),indodicarbocyanine 5 (Cy®5), indodicarbocyanine 5.5 (Cy®5.5),3-(-carboxy-pentyl)-3′-ethyl-5,5′-dimethyloxacarbocyanine (CyA);1H,5H,11H,15H-Xantheno[2,3,4-ij:5,6,7-i′,j′]diquinolizin-18-ium, 9-[2(or4)-[[[6-[2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4(or2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahydro-inner salt (TR or TexasRed®); BODIPY® dyes; benzoxazoles; stilbenes; pyrenes; and the like.

For nucleotide sequencing, certain combinations of luminescently labelednucleotides may be preferred. In some embodiments, at least one of theluminescently labeled nucleotides comprises a cyanine dye, or analogthereof. In some embodiments, at least one luminescently labelednucleotides comprises a rhodamine dye, or analog thereof. In someembodiments, at least two luminescently labeled nucleotides eachcomprise a cyanine dye, or analog thereof. In some embodiments, at leasttwo luminescently labeled nucleotides each comprise a rhodamine dye, oranalog thereof. In some embodiments, at least three luminescentlylabeled nucleotides each comprise a cyanine dye, or analog thereof. Insome embodiments, at least three luminescently labeled nucleotides eachcomprise a rhodamine dye, or analog thereof. In some embodiments, atleast four luminescently labeled nucleotides each comprise a cyaninedye, or analog thereof. In some embodiments, at least four luminescentlylabeled nucleotides each comprise a rhodamine dye, or analog thereof. Insome embodiments, three luminescently labeled nucleotides comprise acyanine dye, or analog thereof, and a fourth luminescently labelednucleotide comprises a rhodamine dye, or analog thereof. In someembodiments, two luminescently labeled nucleotides comprise a cyaninedye, or analog thereof, and a third, and optionally a fourth,luminescently labeled nucleotide comprises a rhodamine dye, or analogthereof. In some embodiments, three luminescently labeled nucleotidescomprise a rhodamine dye, or analog thereof, and a third, and optionallya fourth, luminescently labeled nucleotide comprises a cyanine dye, oranalog thereof.

In some embodiments, at least one labeled nucleotides is linked to twoor more dyes (e.g., two or more copies of the same dye and/or two ormore different dyes).

In some embodiments, at least two luminescently labeled nucleotidesabsorb a first excitation energy, wherein at least one of theluminescently labeled nucleotides comprises a cyanine dye, or analogthereof, and at least one of the luminescently labeled nucleotidescomprises a rhodamine dye, or an analog thereof. In some embodiments, atleast two luminescently labeled nucleotides absorb a second excitationenergy, wherein at least one of the luminescently labeled nucleotidescomprises a cyanine dye, or analog thereof, and at least one of theluminescently labeled nucleotides comprises a rhodamine dye, or ananalog thereof. In some embodiments, at least two luminescently labelednucleotides absorb a first excitation energy, wherein at least one ofthe luminescently labeled nucleotides comprises a cyanine dye, or analogthereof, and at least one of the luminescently labeled nucleotidescomprises a rhodamine dye, or an analog thereof, and at least twoadditional luminescently labeled nucleotides absorb a second excitationenergy, wherein at least one of the luminescently labeled nucleotidescomprises a cyanine dye, or analog thereof, and at least one of theluminescently labeled nucleotides comprises a rhodamine dye, or ananalog thereof.

In some embodiments, at least two luminescently labeled nucleotidesabsorb a first excitation energy, wherein at least one of theluminescently labeled nucleotides has a luminescent lifetime of lessthan about 1 ns, and at least one of the luminescently labelednucleotides has a luminescent lifetime of greater than 1 ns. In someembodiments, at least two luminescently labeled nucleotides absorb asecond excitation energy, wherein at least one of the luminescentlylabeled nucleotides has a luminescent lifetime of less than about 1 ns,and at least one of the luminescently labeled nucleotides has aluminescent lifetime of greater than 1 ns. In some embodiments, at leasttwo luminescently labeled nucleotides absorb a first excitation energy,wherein at least one of the luminescently labeled nucleotides has aluminescent lifetime of less than about 1 ns, and at least one of theluminescently labeled nucleotides has a luminescent lifetime of greaterthan 1 ns, and at least additional two luminescently labeled nucleotidesabsorb a second excitation energy, wherein at least one of theluminescently labeled nucleotides has a luminescent lifetime of lessthan about 1 ns, and at least one of the luminescently labelednucleotides has a luminescent lifetime of greater than 1 ns.

In certain embodiments, the luminescent label is a dye selected fromTable 1. The dyes listed in Table 1 are non-limiting, and theluminescent labels of the application may include dyes not listed inTable 1. In certain embodiments, the luminescent labels of one or moreluminescently labeled nucleotides is selected from Table 1. In certainembodiments, the luminescent labels of four or more luminescentlylabeled nucleotides is selected from Table 1.

TABLE 1 Exemplary fluorophores. Fluorophores 5/6-Carboxyrhodamine 6GChromis 678C DyLight ® 655-B1 5-Carboxyrhodamine 6G Chromis 678ZDyLight ® 655-B2 6-Carboxyrhodamine 6G Chromis 770A DyLight ® 655-B36-TAMRA Chromis 770C DyLight ® 655-B4 Alexa Fluor ® 350 Chromis 800ADyLight ® 662Q Alexa Fluor ® 405 Chromis 800C DyLight ® 675-B1 AlexaFluor ® 430 Chromis 830A DyLight ® 675-B2 Alexa Fluor ® 480 Chromis 830CDyLight ® 675-B3 Alexa Fluor ® 488 Cy ®3 DyLight ® 675-B4 Alexa Fluor ®514 Cy ®3.5 DyLight ® 679-C5 Alexa Fluor ® 532 Cy ®3B DyLight ® 680Alexa Fluor ® 546 Cy ®5 DyLight ® 683Q Alexa Fluor ® 555 Dyomics-350DyLight ® 690-B1 Alexa Fluor ® 568 Dyomics-350XL DyLight ® 690-B2 AlexaFluor ® 594 Dyomics-360XL DyLight ® 696Q Alexa Fluor ® 610-XDyomics-370XL DyLight ® 700-B1 Alexa Fluor ® 633 Dyomics-375XL DyLight ®700-B1 Alexa Fluor ® 647 Dyomics-380XL DyLight ® 730-B1 Alexa Fluor ®660 Dyomics-390XL DyLight ® 730-B2 Alexa Fluor ® 680 Dyomics-405DyLight ® 730-B3 Alexa Fluor ® 700 Dyomics-415 DyLight ® 730-B4 AlexaFluor ® 750 Dyomics-430 DyLight ® 747 Alexa Fluor ® 790 Dyomics-431DyLight ® 747-B1 AMCA Dyomics-478 DyLight ® 747-B2 ATTO 390Dyomics-480XL DyLight ® 747-B3 ATTO 425 Dyomics-481XL DyLight ® 747-B4ATTO 465 Dyomics-485XL DyLight ® 755 ATTO 488 Dyomics-490 DyLight ® 766QATTO 495 Dyomics-495 DyLight ® 775-B2 ATTO 514 Dyomics-505 DyLight ®775-B3 ATTO 520 Dyomics-510XL DyLight ® 775-B4 ATTO 532 Dyomics-511XLDyLight ® 780-B1 ATTO 542 Dyomics-520XL DyLight ® 780-B2 ATTO 550Dyomics-521XL DyLight ® 780-B3 ATTO 565 Dyomics-530 DyLight ® 800 ATTO590 Dyomics-547 DyLight ® 830-B2 ATTO 610 Dyomics-547P1 eFluor ® 450ATTO 620 Dyomics-548 Eosin ATTO 633 Dyomics-549 FITC ATTO 647Dyomics-549P1 Fluorescein ATTO 647N Dyomics-550 HiLyte ™ Fluor 405 ATTO655 Dyomics-554 HiLyte ™ Fluor 488 ATTO 665 Dyomics-555 HiLyte ™ Fluor532 ATTO 680 Dyomics-556 HiLyte ™ Fluor 555 ATTO 700 Dyomics-560HiLyte ™ Fluor 594 ATTO 725 Dyomics-590 HiLyte ™ Fluor 647 ATTO 740Dyomics-591 HiLyte ™ Fluor 680 ATTO Oxa12 Dyomics-594 HiLyte ™ Fluor 750ATTO Rho101 Dyomics-601XL IRDye ® 680LT ATTO Rho11 Dyomics-605 IRDye ®750 ATTO Rho12 Dyomics-610 IRDye ® 800CW ATTO Rho13 Dyomics-615 JOE ATTORho14 Dyomics-630 LightCycler ® 640R ATTO Rho3B Dyomics-631LightCycler ® Red 610 ATTO Rho6G Dyomics-632 LightCycler ® Red 640 ATTOThio12 Dyomics-633 LightCycler ® Red 670 BD Horizon ™ V450 Dyomics-634LightCycler ® Red 705 BODIPY ® 493/501 Dyomics-635 Lissamine Rhodamine BBODIPY ® 530/550 Dyomics-636 Napthofluorescein BODIPY ® 558/568Dyomics-647 Oregon Green ® 488 BODIPY ® 564/570 Dyomics-647P1 OregonGreen ® 514 BODIPY ® 576/589 Dyomics-648 Pacific Blue ™ BODIPY ® 581/591Dyomics-648P1 Pacific Green ™ BODIPY ® 630/650 Dyomics-649 PacificOrange ™ BODIPY ® 650/665 Dyomics-649P1 PET BODIPY ® FL Dyomics-650PF350 BODIPY ® FL-X Dyomics-651 PF405 BODIPY ® R6G Dyomics-652 PF415BODIPY ® TMR Dyomics-654 PF488 BODIPY ® TR Dyomics-675 PF505 C5.5Dyomics-676 PF532 C7 Dyomics-677 PF546 CAL Fluor ® Gold 540 Dyomics-678PF555P CAL Fluor ® Green 510 Dyomics-679P1 PF568 CAL Fluor ® Orange 560Dyomics-680 PF594 CAL Fluor ® Red 590 Dyomics-681 PF610 CAL Fluor ® Red610 Dyomics-682 PF633P CAL Fluor ® Red 615 Dyomics-700 PF647P CALFluor ® Red 635 Dyomics-701 Quasar ® 570 Cascade ® Blue Dyomics-703Quasar ® 670 CF ™350 Dyomics-704 Quasar ® 705 CF ™405M Dyomics-730Rhoadmine 123 CF ™405S Dyomics-731 Rhodamine 6G CF ™488A Dyomics-732Rhodamine B CF ™514 Dyomics-734 Rhodamine Green CF ™532 Dyomics-749Rhodamine Green-X CF ™543 Dyomics-749P1 Rhodamine Red CF ™546Dyomics-750 ROX CF ™555 Dyomics-751 ROX CF ™568 Dyomics-752 Seta ™ 375CF ™594 Dyomics-754 Seta ™ 470 CF ™620R Dyomics-776 Seta ™ 555 CF ™633Dyomics-777 Seta ™ 632 CF ™633-V1 Dyomics-778 Seta ™ 633 CF ™640RDyomics-780 Seta ™ 650 CF ™640R-V1 Dyomics-781 Seta ™ 660 CF ™640R-V2Dyomics-782 Seta ™ 670 CF ™660C Dyomics-800 Seta ™ 680 CF ™660RDyomics-831 Seta ™ 700 CF ™680 DyLight ® 350 Seta ™ 750 CF ™680RDyLight ® 405 Seta ™ 780 CF ™680R-V1 DyLight ® 415-Co1 Seta ™ APC-780CF ™750 DyLight ® 425Q Seta ™ PerCP-680 CF ™770 DyLight ® 485-LS Seta ™R-PE-670 CF ™790 DyLight ® 488 Seta ™646 Chromeo ™ 642 DyLight ® 504QSeta ™u 380 Chromis 425N DyLight ® 510-LS Seta ™u 425 Chromis 500NDyLight ® 515-LS Seta ™u 647 Chromis 515N DyLight ® 521-LS Seta ™u 405Chromis 530N DyLight ® 530-R2 Sulforhodamine 101 Chromis 550A DyLight ®543Q TAMRA Chromis 550C DyLight ® 550 TET Chromis 550Z DyLight ® 554-R0Texas Red ® Chromis 560N DyLight ® 554-R1 TMR Chromis 570N DyLight ®590-R2 TRITC Chromis 577N DyLight ® 594 Yakima Yellow ™ Chromis 600NDyLight ® 610-B1 Zenon ® Chromis 630N DyLight ® 615-B2 Zy3 Chromis 645ADyLight ® 633 Zy5 Chromis 645C DyLight ® 633-B1 Zy5.5 Chromis 645ZDyLight ® 633-B2 Zy7 Chromis 678A DyLight ® 650 Abberior ® ® Star 635Square 635 Square 650 Square 660 Square 672 Square 680 Abberior ® Star440SXP Abberior ® Star 470SXP Abberior ® Star 488 Abberior ® Star 512Abberior ® Star 520SXP Abberior ® Star 580 Abberior ® Star 600Abberior ® Star 635 Abberior ® Star 635P Abberior ® Star RED

Dyes may also be classified based on the wavelength of maximumabsorbance or emitted luminescence. Table 2 provides exemplaryfluorophores grouped into columns according to approximate wavelength ofmaximum absorbance. The dyes listed in Table 2 are non-limiting, and theluminescent labels of the application may include dyes not listed inTable 2. The exact maximum absorbance or emission wavelength may notcorrespond to the indicated spectral ranges. In certain, embodiments,the luminescent labels of one or more luminescently labeled nucleotidesis selected from the “Red” group listed in Table 2. In certainembodiments, the luminescent labels of one or more luminescently labelednucleotides is selected from the “Green” group listed in Table 2. Incertain embodiments, the luminescent labels of one or more luminescentlylabeled nucleotides is selected from the “Yellow/Orange” group listed inTable 2. In certain embodiments, the luminescent labels of fournucleotides are selected such that all are selected from one of the“Red”, “Yellow/Orange”, or “Green” group listed in Table 2. In certainembodiments, the luminescent labels of four nucleotides are selectedsuch that three are selected from a first group of the “Red”,“Yellow/Orange”, and “Green” groups listed in Table 2, and the fourth isselected from a second group of the “Red”, “Yellow/Orange”, and “Green”groups listed in Table 2. In certain embodiments, the luminescent labelsof four nucleotides are selected such that two are selected from a firstof the “Red”, “Yellow/Orange”, and “Green” group listed in Table 2, andthe third and fourth are selected from a second group of the “Red”,“Yellow/Orange”, and “Green” groups listed in Table 2. In certainembodiments, the luminescent labels of four nucleotides are selectedsuch that two are selected from a first of the “Red”, “Yellow/Orange”,and “Green” groups listed in Table 2, and a third is selected from asecond group of the “Red”, “Yellow/Orange”, and “Green” groups listed inTable 2, and a fourth is selected from a third group of the “Red”,“Yellow/Orange”, and “Green” groups listed in Table 2.

TABLE 2 Exemplary fluorophores by spectral range. “Green” 520-570 nm“Yellow/Orange” 570-620 nm “Red” 620-670 nm 5/6-Carboxyrhoadmine 6GAlexa Fluor ® 594 Alexa Fluor ® 633 6-TAMRA Alexa Fluor ® 610-X AlexaFluor ® 647 Alexa Fluor ® 532 ATTO 590 Alexa Fluor ® 660 Alexa Fluor ®546 ATTO 610 ATTO 633 Alexa Fluor ® 555 ATTO 620 ATTO 647 Alexa Fluor ®568 BODIPY ® 576/589 ATTO 647N ATTO 520 BODIPY ® 581/591 ATTO 655 ATTO532 CF ™594 ATTO 665 ATTO 542 CF ™620R ATTO 680 ATTO 550 Chromis 570NATTO Rho14 ATTO 565 Chromis 577N BODIPY ® 630/650 BODIPY ® 530/550Chromis 600N BODIPY ® 650/665 BODIPY ® 558/568 Dyomics-590 CAL Fluor ®Red 635 BODIPY ® 564/570 Dyomics-591 CF ™ 633-V1 CF ™514 Dyomics-594CF ™ 640R-V1 CF ™532 Dyomics-601XL CF ™633 CF ™543 Dyomics-605 CF ™640RCF ™546 Dyomics-610 CF ™640R-V2 CF ™555 Dyomics-615 CF ™660C CF ™568DyLight ® 590-R2 CF ™660R Chromis 530N DyLight ® 594 CF ™680 Chromis550A DyLight ® 610-B1 CF ™680R Chromis 550C DyLight ® 615-B2 CF ™680R-V1Chromis 550Z HiLyte ™ Fluor 594 Chromeo ™ 642 Chromis 560NLightCycler ® ® Red 610 Chromis 630N Cy ®3 PF594 Chromis 645A Cy ®3.5PF594 Chromis 645A Cy ®3B PF610 Chromis 645C Dyomics-530 Quasar ® 570Chromis 645Z Dyomics-547 Abberior ® Star 580 Cy ®5 Dyomics-547P1Abberior ® Star 600 Cy ®5.5 Dyomics-548 Dyomics-630 Dyomics-549P1Dyomics-631 Dyomics-550 Dyomics-632 Dyomics-554 Dyomics-633 Dyomics-555Dyomics-634 Dyomics-556 Dyomics-635 Dyomics-560 Dyomics-636 DyLight ®521-LS Dyomics-647 DyLight ® 530-R2 Dyomics-647P1 DyLight ® 543QDyomics-648 DyLight ® 550 Dyomics-648P1 DyLight ® 554-R0 Dyomics-649DyLight ® 554-R1 Dyomics-649P1 HiLyte ™ Fluor 532 Dyomics-650 HiLyte ™Fluor 555 Dyomics-651 PF532 Dyomics-652 PF546 Dyomics-654 PF555PDyLight ® 633 PF568 DyLight ® 633-B1 Seta ™ 555 DyLight ® 633-B2Abberior ® Star 520SXP DyLight ® 650 DyLight ® 655-B1 DyLight ® 655-B2DyLight ® 655-B3 DyLight ® 655-B4 DyLight ® 662Q DyLight ® 680 DyLight ®683Q HiLyte ™ Fluor 647 HiLyte ™ Fluor 680 LightCycler ® ® 640RLightCycler ® Red 640 LightCycler ® Red 670 PF633P PF647P Quasar ® 670Seta ™ 632 Seta ™ 633 Seta ™ 650 Seta ™ 660 Seta ™ 670 Seta ™Tau 647Square 635 Square 650 Square 660 Abberior ® Star 635 Abberior ® Star635P Abberior ® Star RED

In certain embodiments, the luminescent label may be (Dye 101), (Dye102), (Dye 103), (Dye 104), (Dye 105), or (Dye 106), of formulae (in NHSester form):

or an analog thereof. In some embodiments, each sulfonate or carboxylateis independently optionally protonated. In some embodiments, the dyesabove are attached to the linker or nucleotide by formation of an amidebond at the indicated point of attachment.

In certain embodiments, the luminescent label may comprise a first andsecond chromophore. In some embodiments, an excited state of the firstchromophore is capable of relaxation via an energy transfer to thesecond chromophore. In some embodiments, the energy transfer is aFörster resonance energy transfer (FRET). Such a FRET pair may be usefulfor providing a luminescent label with properties that make the labeleasier to differentiate from amongst a plurality of luminescent labels.In certain embodiments, the FRET pair may absorb excitation energy in afirst spectral range and emit luminescence in a second spectral range.

For a set of luminescently labeled molecules (e.g., luminescentlylabeled nucleotides), the properties of a luminescently labeled FRETpair may allow for selection of a plurality of distinguishable molecules(e.g., nucleotides). In some embodiments, the second chromophore of aFRET pair has a luminescent lifetime distinct from a plurality of otherluminescently labeled molecules. In some embodiments, the secondchromophore of a FRET pair has a luminescent intensity distinct from aplurality of other luminescently labeled molecules. In some embodiments,the second chromophore of a FRET pair has a luminescent lifetime andluminescent intensity distinct from a plurality of other luminescentlylabeled molecules. In some embodiments, the second chromophore of a FRETpair emits photons in a spectral range distinct from a plurality ofother luminescently labeled molecules. In some embodiments, the firstchromophore of a FRET pair has a luminescent lifetime distinct from aplurality of luminescently labeled molecules. In certain embodiments,the FRET pair may absorb excitation energy in a spectral range distinctfrom a plurality of other luminescently labeled molecules. In certainembodiments, the FRET pair may absorb excitation energy in the samespectral range as one or more of a plurality of other luminescentlylabeled molecules.

In some embodiments, two or more nucleotides can be connected to aluminescent label, wherein the nucleotides are connected to distinctlocations on the luminescent label. A non-limiting example could includea luminescent molecule that contains two independent reactive chemicalmoieties (e.g., azido group, acetylene group, carboxyl group, aminogroup) that are compatible with a reactive moiety on a nucleotideanalog. In such an embodiment, a luminescent label could be connected totwo nucleotide molecules via independent linkages. In some embodiments,a luminescent label can comprise two or more independent connections totwo or more nucleotides.

In some embodiments, two or more nucleotides can be connected to aluminescent dye via a linker (e.g., a branched linker or a linker withtwo or more reactive sites onto which nucleotides and/or dyes can beattached). Accordingly, in some embodiments, two or more nucleotides(e.g., of the same type) can be linked to two or more dyes (e.g., of thesame type).

In some embodiments, a luminescent label can comprise a quantum dot withluminescent properties. In some embodiments, one or more nucleotides areconnected to a quantum dot. In some embodiments, one or more nucleotidesare connected to a quantum dot via connections to distinct sites of theprotein. In some embodiments, the surface of a quantum dot is coatedwith nucleotide molecules. In certain embodiments, a quantum dot iscovalently connected to one or more nucleotides (e.g., via reactivemoieties on each component). In certain embodiments, a quantum dot isnon-covalently connected to one or more nucleotides (e.g., viacompatible non-covalent binding partners on each component). In someembodiments, the surface of a quantum dot comprises one or morestreptavidin molecules that are non-covalently bound to one or morebiotinylated nucleotides.

In some embodiments, a luminescent label can comprise a protein withluminescent properties. In some embodiments, one or more nucleotides areconnected to a luminescent protein. In some embodiments, one or morenucleotides are connected to a luminescent protein via connections todistinct sites of the protein. In certain embodiments, the luminescentlabels of four nucleotides are selected such that one nucleotide islabeled with a fluorescent protein while the remaining three nucleotidesare labeled with fluorescent dyes (e.g., the non-limiting examples inTables 1 and 2). In certain embodiments, the luminescent labels of fournucleotides are selected such that two nucleotides are labeled withfluorescent proteins while the remaining two nucleotides are labeledwith fluorescent dyes (e.g., the non-limiting examples in Tables 1 and2). In certain embodiments, the luminescent labels of four nucleotidesare selected such that three nucleotides are labeled with fluorescentproteins while the remaining nucleotide is labeled with a fluorescentdye (e.g., the non-limiting examples in Tables 1 and 2). In someembodiments, the luminescent labels of four nucleotides are selectedsuch that all four nucleotides are labeled with fluorescent proteins.

According to some aspects of the application, luminescent labels (e.g.,dyes, for example fluorophores) can damage polymerases in a sequencingreaction that is exposed to excitation light. In some aspects, thisdamage occurs during the incorporation of a luminescently labelednucleotide, when the luminescent molecule is held in close proximity tothe polymerase enzyme. Non-limiting examples of damaging reactionsinclude the formation of a covalent bond between the polymerase andluminescent molecule and emission of radiative or non-radiative decayfrom the luminescent molecule to the enzyme. This can shorten theeffectiveness of the polymerase and reduce the length of a sequencingrun.

In some embodiments, a nucleotide and a luminescent label are connectedby a relatively long linker or linker configuration to keep theluminescent label away from the polymerase during incorporation of thelabeled nucleotide. The term “linker configuration” is used herein torefer to the entire structure connecting the luminescent molecule(s) tothe nucleotide(s) and does not encompass the luminescent molecule(s) orthe nucleotide(s).

In some embodiments, a single linker connects a luminescent molecule toa nucleotide. In some embodiments, a linker contains one or more pointsof divergence so that two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) nucleotides are connected to each luminescent molecule, two ormore (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) luminescent moleculesare connected to each nucleotide, or two or more (e.g., 2, 3, 4, 5, 6,7, 8, 9, 10, or more) nucleotides are connected to two or more (e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, or more) luminescent molecules.

In some embodiments, the linker configuration determines the distancebetween the luminescent label and the nucleotide. In some embodiments,the distance is about 1 nm or 2 nm to about 20 nm. For example, morethan 2 nm, more than 5 nm, 5-10 nm, more than 10 nm, 10-15 nm, more than15 nm, 15-20 nm, more than 20 nm. However, the distance between theluminescent label and the nucleotide cannot be too long since theluminescent label needs to be within the illumination volume to beexcited when the nucleotide is held within the active site of theenzyme. Accordingly, in some embodiments, the overall linker length isless than 30 nm, less than 25 nm, around 20 nm, or less than 20 nm.

In some embodiments, a protecting molecule is included within a linkerconfiguration. A protecting molecule can be a protein, proteinhomodimer, protein heterodimer, protein oligomer, a polymer, or othermolecule that can protect the polymerase from the damaging reactionsthat can occur between the enzyme and the luminescent label.Non-limiting examples of protecting molecules include proteins (e.g.,avidin, streptavidin, Traptavidin, NeutrAvidin, ubiquitin), proteincomplexes (e.g., Trypsin:BPTI, barnase:barstar, colicin E9 nuclease:Im9immunity protein), nucleic acids (e.g., deoxyribonucleic acid,ribonucleic acid), polysaccharides, lipids, and carbon nanotubes.

In some embodiments, the protecting molecule is an oligonucleotide(e.g., a DNA oligonucleotide, an RNA oligonucleotide, or a variantthereof). In some embodiments, the oligonucleotide is single-stranded.In some embodiments, a luminescent label is attached directly orindirectly to one end of the single-stranded oligonucleotide (e.g., the5′ end or the 3′ end) and one or more nucleotides are attached directlyor indirectly to the other end of the single-stranded oligonucleotide(e.g., the 3′ end or the 5′ end). For example, the single-strandedoligonucleotide can comprise a luminescent label attached to the 5′ endof the oligonucleotide and one or more nucleotides attached to the 3′end of the oligonucleotide. In some embodiments, the oligonucleotide isdouble-stranded (e.g., the oligonucleotide comprises two annealed,complementary oligonucleotide strands). In some embodiments, aluminescent label is attached directly or indirectly to one end of thedouble-stranded oligonucleotide and one or more nucleotides are attacheddirectly or indirectly to the other end of the double-strandednucleotide. In some embodiments, a luminescent label is attacheddirectly or indirectly to one strand of the double-strandedoligonucleotide and one or more nucleotides are attached directly orindirectly to the other strand of the double-stranded nucleotide. Forexample, the double-stranded oligonucleotide can comprise a luminescentlabel attached to the 5′ end of one strand of the oligonucleotide andone or more nucleotides attached to the 5′ end of the other strand.

In some embodiments, a protecting molecule is connected to one or moreluminescent molecules and to one or more nucleotide molecules. In someembodiments, the luminescent molecule(s) are not adjacent to thenucleotide(s). For example, one or more luminescent molecules can beconnected on a first side of the protecting molecule and one or morenucleotides can be connected to a second side of the protectingmolecule, wherein the first and second sides of the protecting moleculeare distant from each other. In some embodiments, they are onapproximately opposite sides of the protecting molecule.

The distance between the point at which a protecting molecule isconnected to a luminescent label and the point at which the protectingmolecule is connected to a nucleotide can be a linear measurementthrough space or a non-linear measurement across the surface of theprotecting molecule. The distance between the luminescent label andnucleotide connection points on a protecting molecule can be measured bymodeling the three-dimensional structure of the protecting molecule. Insome embodiments, this distance can be 2, 4, 6, 8, 10, 12, 14, 16, 18,20 nm or more. Alternatively, the relative positions of the luminescentlabel and nucleotide on a protecting molecule can be described bytreating the structure of the protecting molecule as a quadratic surface(e.g., ellipsoid, elliptic cylinder). In some embodiments, theluminescent label and the nucleotide are separated by a distance that isat least one eighth of the distance around an ellipsoidal shaperepresenting the protecting molecule. In some embodiments, theluminescent label and the nucleotide are separated by a distance that isat least one quarter of the distance around an ellipsoidal shaperepresenting the protecting molecule. In some embodiments, theluminescent label and the nucleotide are separated by a distance that isat least one third of the distance around an ellipsoidal shaperepresenting the protecting molecule. In some embodiments, theluminescent label and the nucleotide are separated by a distance that isone half of the distance around an ellipsoidal shape representing theprotecting molecule.

FIG. 10 illustrates a non-limiting example 10-1 of a luminescentmolecule (200) separated from a nucleotide (250) by a protectingmolecule (100). The luminescent molecule (200) is connected to theprotecting molecule (100) via a linker (300), wherein the linker (300)is attached to the protecting molecule (100). The nucleotide (250) isconnected to the protecting molecule (100) via a linker (350), whereinthe linker (350) is attached to the protecting molecule (100).Protecting molecules can be useful to provide a steric barrier thatprevents a luminescent label from getting near the polymerase.Protecting molecules can be useful to absorb, or protect the polymerasefrom, radiative and non-radiative decay emitted by a luminescentmolecule. Protecting molecules can be useful to provide both a stericbarrier and a decay barrier between the luminescent label and thepolymerase.

The size of a protecting molecule should be such that a luminescentlabel is unable or unlikely to directly contact the polymerase when anucleotide is held within the active site of the enzyme. The size of aprotecting molecule should also be such that an attached luminescentlabel is within the illumination volume to be excited when a nucleotideis held within the active site of the enzyme. The size of a protectingmolecule should be chosen with consideration to the linker that isselected to connect a luminescent label to the protecting molecule andthe linker that is selected to connect a nucleotide to the protectingmolecule. The protecting molecule and the linkers used to connect theluminescent label and nucleotide (e.g., nucleoside polyphosphate)comprise the linker configuration, wherein the size of the linkerconfiguration should be such that the luminescent label is unable todirectly contact the polymerase when the nucleotide is held within theactive site of the enzyme.

The protecting molecule (and/or the linker configuration comprising theprotecting molecule) is preferably water soluble. In some embodiments,it is preferable that the protecting molecule (and/or the linkerconfiguration comprising the protecting molecule) has an net negativecharge.

In some embodiments, the label (e.g., the luminescent molecule) is notcovalently linked to the nucleotide due to one or more non-covalentlinkages connecting the label to the nucleotide. In some embodiments,one or more linkers are non-covalently attached to the protectingmolecule and/or the luminescent molecule(s). In some embodiments, one ormore linkers are non-covalently attached to the protecting moleculeand/or the nucleotides. In some embodiments, a luminescent label iscovalently attached to a linker, wherein the linker is non-covalentlyattached to the protecting molecule (e.g., via one or more bindingpartners). In some embodiments, a nucleotide is covalently attached to alinker, wherein the linker is non-covalently attached to the protectingmolecule (e.g., via one or more binding partners).

FIGS. 10-15 illustrate non-limiting examples of one or more luminescentmolecules and one or more nucleotides connected via a protectingmolecule, wherein one or more luminescent molecules and one or morenucleotides are non-covalently attached to the protecting molecule.

FIG. 10 illustrates a non-limiting example 10-2 that comprises theelements of 10-1, wherein a linker comprises a non-covalent bindingligand (400) that non-covalently attaches a luminescent molecule to anon-covalent binding site (110) of a protecting molecule (shaded shape)and a second linker comprises a non-covalent binding ligand (450) thatnon-covalently attaches a nucleotide to a second non-covalent bindingsite (120) of the protecting molecule.

FIG. 10 illustrates a non-limiting example 10-3 that comprises theelements of 10-2, wherein a protecting molecule (shaded shape) comprisesfour independent non-covalent binding sites. A second independentluminescent molecule can be non-covalently attached to a non-covalentbinding site (111) of the protecting molecule via a non-covalent bindingligand (401). A second independent nucleotide molecule can benon-covalently attached to a non-covalent binding site (121) of theprotecting molecule via a non-covalent binding ligand (451). In thisembodiment depicting two independent luminescent molecules and twoindependent nucleotides non-covalently bound at four independentnon-covalent binding sites of a protecting molecule, the number ofluminescent molecules and nucleotides were arbitrarily chosen. In someembodiments, the four independent non-covalent binding sites of aprotecting molecule comprise one luminescent molecule at one site andone nucleotide in each of the remaining three sites. In someembodiments, the four independent non-covalent binding sites of aprotecting molecule comprise one nucleotide at one site and oneluminescent molecule in each of the remaining three sites.

FIG. 10 illustrates a non-limiting example 10-4 that comprises theelements of 10-3, wherein a linker (301) comprises two independentnon-covalent binding ligands (400, 401) that non-covalently attach asingle luminescent molecule via two independent non-covalent bindingsites of a protecting molecule (shaded shape) and a linker (351)comprises two independent non-covalent binding ligands (450, 451) thatnon-covalently attach a single nucleotide via the remaining twoindependent non-covalent binding sites of the protecting molecule.

FIG. 11 illustrates a non-limiting example 11-1 that comprises theelements of 10-4, wherein the linker (301) comprises a point ofdivergence (302) that permits the attachment of two luminescentmolecules.

FIG. 11 illustrates a non-limiting example 11-2 that comprises theelements of 11-1, wherein the linker (351) comprises a point ofdivergence (352) that permits the attachment of two nucleotides.

FIG. 11 illustrates a non-limiting example 11-3 that comprises theelements of 11-2, wherein the linker (302) comprises two additionalpoints of divergence (304) that permit the attachment of fourluminescent molecules and the linker (352) comprises two additionalpoints of divergence (354) that permit the attachment of fournucleotides.

FIG. 11 illustrates a non-limiting example 11-4 that comprises theelements of 11-3, wherein each point of divergence depicted in (304)comprises an additional attachment moiety that permits the attachment ofan additional luminescent molecule for a total of six luminescentmolecules and each point of divergence depicted in (354) comprises anadditional attachment moiety that permits the attachment of anadditional nucleotide for a total of six nucleotides.

FIG. 12 illustrates a non-limiting example of a set of labelednucleotide molecules that can be used in a sequencing reaction: thymine12-1, adenine 12-2, cytosine 12-3, and guanine 12-4. In this embodiment,a protecting molecule comprising four non-covalent binding sites is usedto connect six of the same type of nucleotide to one or more luminescentmolecules. In this embodiment, six of the same type of nucleotide areattached to the protecting molecule via two separate linkers, eachlinker comprising an independent non-covalent binding ligand that iscovalently linked to three of the same type of nucleotide. As depictedby 12-1, 12-2, and 12-3, a single luminescent molecule is non-covalentlyattached to the protecting molecule via a linker comprising twoindependent non-covalent binding ligands that non-covalently attach totwo independent binding sites of the protecting molecule. As depicted in12-4, a point of divergence in the luminescent linker permits theaddition of a second luminescent molecule.

In a sequencing experiment comprising the four exemplary nucleotides,the luminescent labels of 12-1, 12-2, and 12-3 can comprise three uniqueluminescent molecules (e.g., three different fluorophores). Since theseexemplary molecules each contain only a single fluorophore, theproperties (e.g., luminescent lifetime, luminescent intensity, emissionwavelength) used to distinguish amongst thymine, adenine, or cytosineincorporation would be highly similar or indistinguishable if uniquefluorophores are not used. Guanine 12-4 is connected to two luminescentmolecules. In some embodiments, the luminescent label of 12-4 cancomprise two of the same luminescent molecule, wherein the selectedmolecule is different from the fluorophores used in 12-1, 12-2, and12-3. In a sequencing reaction comprising four unique nucleotidesconnected to four unique fluorophores, each fluorophore should have oneor more unique luminescent properties (e.g., luminescent lifetime,intensity, emission wavelength, or a combination of two or more thereof)that allow each one to be distinguished among the plurality. In someembodiments, the two fluorophores of 12-4 can comprise two of the samefluorophore, wherein the selected fluorophore is identical to one of thefluorophores of 12-1, 12-2, and 12-3. In a sequencing reactioncomprising unique nucleotides connected to a differing number of thesame luminescent molecule, the differing number of fluorophores shouldconfer unique properties (e.g., increased luminescent intensity) toallow one nucleotide to be distinguished from among the plurality. Thetwo fluorophores of 12-4 can alternatively comprise a FRET pair.

FIG. 12 further illustrates a non-limiting example of a sequencingexperiment and how unique luminescent properties can be used todistinguish among a plurality of luminescently labeled nucleotides 12-5.The luminescent label connected to each base (thymine, adenine,cytosine, guanine) has luminescent properties (e.g., luminescentlifetime, luminescent intensity, and/or emission wavelength) that alloweach labeled nucleotide to be distinguished from the plurality oflabeled nucleotides. The inclusion of multiple nucleotides of the sametype functions to accelerate incorporation rates in a sequencingreaction.

A sequencing experiment utilizing the luminescently labeled nucleotides12-5 can be conducted in exemplary reaction vessel 12-6. The reactiontakes place in a chamber above the waveguide, which serves as a conduitfor excitation energy, delivering the excitation energy to the sample inthe bottom of the reaction chamber by the evanescent wave from thewaveguide. The aperture blocks light radiating from the waveguide tobulk sample and ambient and/or stray light from the sensor, as well asproviding a fabrication path for the reaction chamber. The reactionchamber is an etched structure that places the sample on the bottom andwithin a region of high excitation from the evanescent wave of thewaveguide. Selective surface chemistry is used to provide the bottom andsidewall of the reaction chamber with different composition, so that thesample can be selectively localized to the bottom of the reactionchamber. An exemplary workflow for surface preparation is shown in FIG.7, which depicts a process comprising, inter alia, processes related toachieving selective surface chemistry. For example, passivation ofsurfaces (e.g., as depicted in the non-limiting embodiment shown in FIG.8) can provide a selective surface substrate that will be useful duringfunctionalization of the surface (e.g., as depicted in the non-limitingembodiment shown in FIG. 9).

The incorporation of a specific nucleotide can be distinguished fromamong four luminescently labeled nucleotides during a sequencingreaction per the exemplary workflow 12-7. Throughout the course of anexperiment, there are two distinct periods: a pulse period and adetection period. During the pulse period, lasting 20 picoseconds, noemission light is collected. Following the pulse period is the detectionperiod, lasting 10 nanoseconds, wherein four time bins capture emissionevents occurring over the detection period (i). A pulse and detectionperiod comprise one cycle. Emission events are continuously binned andaccumulated over the course of 1 million cycles (ii). The overalldistribution of emission events across time bins are representative ofluminescent lifetime and can be used to match a particular set of a datato a known lifetime distribution (iii). In some embodiments, thedistribution of emission events (e.g., luminescent lifetime) does notdistinguish one luminescently labeled base from a plurality of otherlabeled molecules. In addition to the distribution of emission events,the quantity of emission events (e.g., luminescent intensity) can beused to identify a single molecule from a plurality of others.

FIG. 13 illustrates a non-limiting example 13-1 that comprises theelements of 10-4, wherein a non-covalent binding site of the protectingmolecule is engineered to be a non-functioning binding site (461). Insome embodiments, the protecting molecule of 13-1 can comprisestreptavidin. In some embodiments, the binding site is non-functionaldue to an inability to non-covalently bind a biotin molecule. Thenon-limiting embodiment 13-1 depicts a single luminescent moleculeattached to a protecting molecule via two independent non-covalentbinding interactions and six nucleotides attached to the protectingmolecule via a single non-covalent binding interaction. In someembodiments, a protecting molecule with three functional non-covalentbinding sites can comprise one or more luminescent molecules attachedvia a single non-covalent interaction and one or more nucleotidesattached via two independent non-covalent interactions. In someembodiments, a protecting molecule with three functional non-covalentbinding sites can comprise one or more luminescent molecules attachedvia two independent non-covalent interactions and one or morenucleotides attached via a single non-covalent interaction.

FIG. 13 illustrates a non-limiting example 13-2 that comprises theelements of 10-3, wherein two non-covalent binding sites of theprotecting molecule are engineered to be non-functioning binding sites(411, 461). In some embodiments, the protecting molecule of 13-2 cancomprise streptavidin. In some embodiments, the binding sites arenon-functional due to an inability to non-covalently bind a biotinmolecule. The non-limiting embodiment 13-2 depicts a single luminescentmolecule attached to the protecting molecule via a single independentnon-covalent binding interaction and six nucleotides attached to theprotecting molecule via a single non-covalent binding interaction. Insome embodiments, a protecting molecule with two functional non-covalentbinding sites can comprise one or more luminescent molecules attachedvia a single non-covalent interaction and one or more nucleotidesattached via a single non-covalent interaction. In some embodiments, thetwo non-functional non-covalent binding sites are cis binding sites ortrans binding sites, wherein “cis binding sites” are defined as being incloser proximity than “trans binding sites.” FIG. 13 further depicts anon-limiting example 13-3 of the generation of a streptavidin moleculewith two non-functional trans binding sites.

In some embodiments, a linker can be comprised of nucleotides. FIG. 14illustrates a non-limiting exemplary reaction scheme in which aluminescent molecule and a nucleotide are connected via a protectingmolecule, wherein the linkers attaching the luminescent molecule and thenucleotide to the protecting molecule comprise oligonucleotides (e.g.,oligonucleotides comprising DNA, RNA, PNA, or modified forms thereof)attached via divalent linkers. A divalent linker (360) that is comprisedof an oligonucleotide is non-covalently bound to two independentnon-covalent binding sites of a protecting molecule (a). A luminescentmolecule (201) fused to an oligonucleotide complementary to the linker(360) is introduced to generate a luminescently labeled protectingmolecule (b). A second divalent linker (361) that is comprised of anoligonucleotide is introduced to form (c). A nucleotide (251) fused toan oligonucleotide complementary to the linker (361) is introduced togenerate the final product (d).

FIG. 15 illustrates a non-limiting exemplary reaction scheme in which aluminescent molecule is attached to a protecting molecule via annealedcomplementary oligonucleotides, wherein each oligonucleotide strandcontains one non-covalent binding ligand compatible with a binding siteon the protecting molecule. A first oligonucleotide (362) that is fusedto a luminescent molecule and non-covalent binding ligand is annealed(i) to a complementary oligonucleotide (363) that is fused to anon-covalent binding ligand. The annealed product (364) is bound to aprotecting molecule (ii) and purified. The purified luminescentlylabeled protecting molecule is bound with nucleotides (iii) using anytechniques or configurations disclosed herein. FIG. 15 also depicts anexemplary sequencing experiment performed using a nucleotide that waslabeled with a Cy®3 dye using a duplex DNA linker analogous to thenon-limiting example in the reaction scheme.

In some embodiments, one or more linkers are covalently attached to theprotecting molecule and/or the luminescent molecule(s). In someembodiments, one or more linkers are covalently attached to theprotecting molecule and/or the nucleotide(s).

FIGS. 16-20 illustrate non-limiting examples of a luminescent moleculeand a nucleotide connected via a protecting molecule, wherein theluminescent molecule and the nucleotide are covalently attached to theprotecting molecule.

FIG. 16 depicts a reaction scheme 16-1 for generating a genericprotecting molecule (shaded shape), wherein the protecting moleculecomprises two genetically-encoded tags. A genetically-encoded tag (325)is designed to form a covalent attachment to a specific reactive groupfused to a luminescent molecule (i) and a separate genetically-encodedtag (375) is designed to form a covalent attachment to a specificreactive group fused to a nucleotide (ii) to form the final product (c).FIG. 16 further depicts a non-limiting embodiment of a scheme 16-2 forthe covalent attachment of a generic substrate comprising abioorthogonal functional group (star shape) such as an azide, aldehyde,or alkyne, via a genetically-encoded tag (curved line), along withnon-limiting examples 16-3 of genetically-encoded tags and kinetic rateconstants.

FIG. 17 depicts a non-limiting example of a reaction scheme 17-1 forgenerating a generic protecting molecule (shaded shape), wherein theprotecting molecule is a protein that comprises a reactive group at theN-terminus and a reactive group at the C-terminus. A first reactivegroup at a first terminus of the protein (326) is designed to form acovalent attachment to a specific reactive group fused to a luminescentmolecule. A second reactive group of the remaining terminus of theprotein (376) is designed to form a covalent attachment to a specificreactive group fused to a nucleotide. Non-limiting examples 17-2 ofreactive N-terminus and C-terminus groups and the corresponding reactivegroups are further depicted.

FIG. 18 depicts a non-limiting example of a reaction scheme 18-1 forgenerating a generic protecting molecule (shaded shape), wherein theprotecting molecule is a protein that comprises a reactive unnaturalamino acid at one part of the protein and a reactive unnatural aminoacid at another part of the protein. A first reactive unnatural aminoacid at a first part of the protein (327) is designed to form a covalentattachment to a specific reactive group fused to a luminescent molecule.A second reactive unnatural amino acid at a second part of the protein(377) is designed to form a covalent attachment to a specific reactivegroup fused to a nucleotide. FIG. 18 further depicts a generic andnon-limiting scheme 18-2 for the chemical labeling of a target protein.In this example, an unnatural amino acid (e.g., an unnatural amino acidselected from non-limiting examples 18-3) bearing a unique biorthogonalfunctionality is introduced site-specifically into a protein via geneticcode expansion and then chemoselectively labeled with an externallyadded probe.

FIG. 19A is a non-limiting example of a luminescent molecule and anucleotide separated by a non-protein protecting molecule (101).Non-limiting examples of non-protein protecting molecules can includenucleic acid molecules (deoxyribonucleic acid, ribonucleic acid),lipids, and carbon nanotubes. As shown, the luminescent molecule and thenucleotide are attached directly to the non-protein protecting molecule(e.g., covalently attached). In some embodiments, the luminescentmolecule and the nucleotide are attached directly to a contiguous partof the non-protein protecting molecule. For example, in someembodiments, the non-protein protecting molecule is a nucleic acidmolecule, and the luminescent molecule and/or nucleotide are bounddirectly to a nucleotide of the nucleic acid molecule. In someembodiments, the luminescent molecule and/or nucleotide are attached tothe non-protein protecting molecule via a linker that is not acontiguous part of the non-protein protecting molecule. For example, insome embodiments, the non-protein protecting molecule is a nucleic acidmolecule, and the luminescent molecule and/or nucleotide are attached tothe nucleic acid via a linker.

In some embodiments, the luminescent molecule and the nucleotide can beattached to the non-protein protecting molecule via reactive moieties,as depicted in FIG. 19B. In this example, a reactive moiety (550) on theluminescent molecule is covalently attached to the non-proteinprotecting molecule via a corresponding reactive moiety (500) on thenon-protein protecting molecule. A reactive moiety (551) on thenucleotide is covalently attached to the non-protein protecting molecule(101) via a corresponding reactive moiety (501) on the non-proteinprotecting molecule. In some embodiments, the reactive moiety 500 and/orthe reactive moiety 501 are attached directly to a contiguous part ofthe non-protein protecting molecule. In some embodiments, the reactivemoiety 500 and/or the reactive moiety 501 are attached to thenon-protein protecting molecule via a linker that is not a contiguouspart of the non-protein protecting molecule.

In some embodiments, a protecting molecule is comprised of aprotein-protein pair. A protein-protein pair can comprise any set ofpolypeptide binding partners (e.g., protein-receptor, enzyme-inhibitor,antibody-antigen). FIG. 20 depicts a non-limiting embodiment of aprotecting molecule comprised of a protein-protein binding pair. In thisnon-limiting example, one polypeptide (102) of the binding pair isorthogonally labeled with luminescent molecules and a second polypeptide(103) is orthogonally linked to nucleotides. Non-limiting examples ofprotein-protein binding pairs include Trypsin-BPTI, barnase-barstar, andcolicin E9 nuclease-Im9 immunity protein.

FIG. 21 depicts non-limiting examples of linker configurationscomprising one or more luminescent molecules attached to a non-covalentbinding ligand. In an embodiment 21-1, a first linker layer comprising anon-covalent binding ligand (410), spacer linker (310), and reactivemoiety (510) is attached to a luminescent molecule (210) comprising acompatible reactive moiety (211). The reactive moiety of the firstlinker layer can be used to attach directly to a luminescent molecule ornucleotide via a compatible reactive moiety. The reactive moiety of thefirst linker layer can be used to attach a second linker layer via acompatible reactive moiety. In an embodiment 21-2, two luminescentmolecules are attached to the first linker layer via a second linkerlayer, wherein the second linker layer comprises a reactive moietycompatible with the reactive moiety of the first layer, a spacer linkercomprising a point of divergence (312), and two reactive moieties (522)compatible with the reactive moiety of the luminescent molecule. The tworeactive moieties of the second linker layer can be used to attachdirectly to luminescent or nucleotide molecules. The two reactivemoieties of the second linker layer can be used to attach a third linkerlayer to further increase the number of luminescent molecules ornucleotides comprising a linker configuration. In an embodiment 21-3,six luminescent molecules are attached to the second linker layer via athird linker layer bound at each reactive moiety (522), wherein eachthird linker layer comprises a compatible reactive moiety (513), aspacer linker that is tri-functionalized (313), and three reactivemoieties (523) compatible with the reactive moieties of the luminescentmolecules.

Accordingly, a luminescent label may be attached to the moleculedirectly, e.g., by a bond, or may be attached via a linker or a linkerconfiguration. In certain embodiments, the linker comprises one or morephosphates. In some embodiments, a nucleotide is connected to aluminescent label by a linker comprising one or more phosphates. As usedherein, a linker described as having one or more phosphates refers to alinker that comprises one or more phosphates present within the linkerstructure and not directly attached to the one or more phosphates of anucleotide. In some embodiments, a nucleotide is connected to aluminescent label by a linker comprising three or more phosphates. Insome embodiments, a nucleotide is connected to a luminescent label by alinker comprising four or more phosphates.

In certain embodiments, a linker comprises an aliphatic chain. In someembodiments a linker comprises —(CH₂)_(n)—, wherein n is an integer from1 to 20, inclusive. In some embodiments, n is an integer from 1 to 10,inclusive. In certain embodiments, a linker comprises a heteroaliphaticchain. In some embodiments, a linker comprises a polyethylene glycolmoiety. In some embodiments, a linker comprises a polypropylene glycolmoiety. In some embodiments, a linker comprises —(CH₂CH₂O)_(n)—, whereinn is an integer from 1 to 20, inclusive. In some embodiments, a linkercomprises —(CH₂CH₂O)_(n)—, wherein n is an integer from 1 to 10,inclusive. In certain embodiments, a linker comprises —(CH₂CH₂O)₄—. Incertain embodiments, a linker comprises one or more arylenes. In someembodiments, a linker comprises one or more phenylenes (e.g.,para-substituted phenylene). In certain embodiments, a linker comprisesa chiral center. In some embodiments, a linker comprises proline, or aderivative thereof. In some embodiments, a linker comprises a prolinehexamer, or a derivative thereof. In some embodiments, a linkercomprises coumarin, or a derivative thereof. In some embodiments, alinker comprises naphthalene, or a derivative thereof. In someembodiments, a linker comprises anthracene, or a derivative thereof. Insome embodiments, a linker comprises a polyphenylamide, or a derivativethereof. In some embodiments, a linker comprises chromanone, or aderivative thereof. In some embodiments, a linker comprises4-aminopropargyl-L-phenylalanine, or a derivative thereof. In certainembodiments, a linker comprises a polypeptide.

In some embodiments, a linker comprises an oligonucleotide. In someembodiments, a linker comprises two annealed oligonucleotides. In someembodiments, the oligonucleotide or oligonucleotides comprisedeoxyribose nucleotides, ribose nucleotide, or locked ribosenucleotides.

In certain embodiments, a linker comprises a photostabilizer. In someembodiments, the linker is of formula:

wherein P^(S) is a photostabilizer, the position labeled d is attachedto a luminescent label, and the position labeled b is attached to anucleotide. In some embodiments, the position labeled d is attached to aluminescent label by a linker as described herein. In some embodiments,the position labeled b is attached to a nucleotide by a linker asdescribed herein.

In certain embodiments, a linker comprises one or more phosphates, analiphatic chain, a heteroaliphatic chain, and one or more amides (e.g.,—C(═O)NH—). In certain embodiments, a linker comprising one or morephosphates and an aliphatic chain can be synthesized via the exemplaryreaction scheme 22-1 depicted in FIG. 22. Exemplary linker structuresare further depicted in 22-2 and 22-3. In certain embodiments, a linkeris selected from linkers depicted in Table 3. Certain exemplary linkerstructures in Table 3 are shown linked to nucleotides (e.g., nucleosidehexaphosphate) and/or dyes.

TABLE 3 Exemplary linkers or linker/dye combination. Linkers

In certain embodiments, a linker comprises one or more phosphates, analiphatic chain, a heteroaliphatic chain, one or more amides (e.g.,—C(═O)NH—), and one or more biotin moieties. In certain embodiments, abiotinylated linker contains one or more functionalizable, reactivemoieties (e.g., acetylene group, azido group). In certain embodiments, alinker is selected from the non-limiting linkers depicted in Table 4.Certain exemplary linker structures in Table 4 are shown linked tonucleotides (e.g., nucleoside hexaphosphate).

TABLE 4 Exemplary biotinylated linkers. Biotinylated Linkers

In some embodiments, a linker is synthesized from precursors comprisinga first layer, and optionally a second and/or third layer. In someembodiments, a “first layer” contains one or more biotin moieties. Incertain embodiments, the first layer of a linker contains a singlebiotin moiety. In certain embodiments, the first layer of a linkercontains two biotin moieties. In some embodiments, a first layercontains one or more reactive moieties (e.g., azido group, acetylenegroup, carboxyl group, amino group). In some embodiments, the firstlayer of a linker is selected from the structures depicted in Table 5.

TABLE 5 Exemplary first linker layers First Layers

In some embodiments, the first layer of a linker can be synthesizedaccording to the following exemplary reaction scheme:

In further embodiments, the first layer of a linker can be synthesizedaccording to the following exemplary reaction scheme:

In yet further embodiments, the first layer of a linker can besynthesized according to the following exemplary reaction scheme:

In some embodiments, a “second layer” contains one or more reactivemoieties (e.g., azido group, acetylene group, carboxyl group, aminogroup). In some embodiments, a second layer is covalently attached to afirst layer of a linker. In some embodiments, a second layer covalentlyconnects a first layer to a third layer. In some embodiments, a “thirdlayer” contains one or more reactive moieties (e.g., azido group,acetylene group, carboxyl group, amino group). In some embodiments, athird layer is covalently attached to a first layer of a linker. In someembodiments, a third layer is covalently connected to a first layer viaa second layer. In some embodiments, a linker comprises a first andsecond layer. In some embodiments, a linker comprises a first and thirdlayer. In some embodiments, a linker comprises a first, second, andthird layer. In certain embodiments, a second and/or third linker layeris selected from the structures depicted in Table 6.

TABLE 6 Exemplary second and third linker layers. Second and ThirdLayers

In some embodiments, a linker comprising more than a first layer (e.g.,a second layer and/or a third layer) can be synthesized according to thefollowing exemplary reaction scheme:

In some embodiments, a linker comprising at least a second and a thirdlayer can be synthesized according to the following exemplary reactionscheme:

Exemplary luminescent labels with biotinylated linkers are shown inTable 7. It should be appreciated that different luminescent labels canbe substituted in place of the dyes depicted in Table 7.

TABLE 7 Exemplary luminescent labels with biotinylated linkers.

Sample Well (e.g., Nanoaperture) Surface Preparation

In certain embodiments, a method of detecting one or more luminescentlylabeled molecules is performed with the molecules confined in a targetvolume (e.g., a reaction volume). In some embodiments, the target volumeis a region within a sample well (e.g., a nanoaperture). Embodiments ofsample wells (e.g., nanoapertures) and the fabrication of sample wells(e.g., nanoapertures) are described elsewhere herein. In certainembodiments, the sample well (e.g., nanoaperture) comprises a bottomsurface comprising a first material and sidewalls formed by a pluralityof metal or metal oxide layers. In some embodiments, the first materialis a transparent material or glass. In some embodiments, the bottomsurface is flat. In some embodiments, the bottom surface is a curvedwell. In some embodiments, the bottom surface includes a portion of thesidewalls below the sidewalls formed by a plurality of metal or metaloxide layers. In some embodiments, the first material is fused silica orsilicon dioxide. In some embodiments, the plurality of layers eachcomprise a metal (e.g., Al, Ti) or metal oxide (e.g., Al₂O₃, TiO₂, TiN).

Passivation

In embodiments when one or more molecule or complex is immobilized onthe bottom surface it may be desirable to passivate the sidewalls toprevent immobilization on the sidewall surfaces. In some embodiments,the sidewalls are passivated by the steps of: depositing a metal ormetal oxide barrier layer on the sidewall surfaces; and applying acoating to the barrier layer. In some embodiments, the metal oxidebarrier layer comprises aluminum oxide. In some embodiments, the step ofdepositing comprises depositing the metal or metal oxide barrier layeron the sidewall surfaces and the bottom surface. In some embodiments,the step of depositing further comprises etching metal or metal oxidebarrier layer off of the bottom surface.

In some embodiments, the barrier layer coating comprises phosphonategroups. In some embodiments, the barrier layer coating comprisesphosphonate groups with an alkyl chain. In some embodiments, the alkylchain comprises a straight-chain saturated hydrocarbon group having 1 to20 carbon atoms. In some embodiments, the barrier layer coatingcomprises hexylphosphonic acid (HPA). In some embodiments, the barrierlayer coating comprises a polymeric phosphonate. In some embodiments,the barrier layer coating comprises polyvinylphosphonic acid (PVPA). Insome embodiments, the barrier layer coating comprises phosphonate groupswith a substituted alkyl chain. In some embodiments, the alkyl chaincomprises one or more amides. In some embodiments, the alkyl chaincomprises one or more poly(ethylene glycol) chains. In some embodiments,the coating comprises phosphonate groups of formula:

wherein n is an integer between 0 and 100, inclusive, and

is hydrogen or a point of attachment to the surface. In some embodimentsn is an integer between 3 and 20, inclusive. In some embodiments, thebarrier layer coating comprises a mixture of different types ofphosphonate groups. In some embodiments, the barrier layer coatingcomprises a mixture of phosphonate groups comprising poly(ethyleneglycol) chains of different PEG weight.

In certain embodiments, the barrier layer comprises nitrodopa groups. Incertain embodiments, the barrier layer coating comprises groups offormula:

wherein R^(N) is an optionally substituted alkyl chain and

is hydrogen or a point of attachment to the surface. In someembodiments, R^(N) comprises a polymer. In some embodiments, R^(N)comprises a poly(lysine) or a poly(ethylene glycol). In someembodiments, the barrier layer comprises a co-polymer of poly(lysine)comprising lysine monomers, wherein the lysine monomers independentlycomprise PEG, nitrodopa groups, phosphonate groups, or primary amines.In certain embodiments, the barrier layer comprises a polymer of formula(P):

In some embodiments, X is —OMe, a biotin group, phosphonate, or silane.In some embodiments, each of i, j, k, and l is independently an integerbetween 0 and 100, inclusive.

FIG. 8 depicts two methods of passivating a metal oxide surface. In 8-1a metal oxide surface is treated with (2-aminoethyl)phosphonic acid,providing a surface coated with (2-aminoethyl)phosphonate groups. In asecond passivation step, the surface is treated with a poly(ethyleneglycol) NHS ester. Reaction of the NHS esters with the amine groups ofthe surface phosphonate groups forms amide bonds to form a surfacecoated with PEG functionalized phosphonate groups. In 8-2 the metaloxide surface is treated with a PEG functionalized phosphonic acid, toprovide in one step a surface coated with PEG functionalized phosphonategroups. The exemplary embodiment 8-2 further depicts the addition of asecond PEG functionalized phosphonic acid, to provide a surface coatedwith two types of PEG functionalized phosphonate groups. In thisexample, the first PEG phosphonic acid with average molecular weight of550, and the second PEG phosphonic acid with average molecular weight of180. As depicted, the shorter PEG chain phosphonic acids may help fillin gaps in the surface coating that remain after treatment with theinitial PEG phosphonic acid with longer PEG chains.

Polymerase Immobilization

In embodiments when one or more molecule or complex is immobilized onthe bottom surface it may be desirable to functionalize the bottomsurface to allow for attachment of one or more molecule or complex. Incertain embodiments, the bottom surface comprises a transparent glass.In certain embodiments, the bottom surface comprises fused silica orsilicon dioxide. In some embodiments, the bottom surface isfunctionalized with a silane. In some embodiments, the bottom surface isfunctionalized with an ionically charged polymer. In some embodiments,the ionically charged polymer comprises poly(lysine). In someembodiments, the bottom surface is functionalized withpoly(lysine)-graft-poly(ethylene glycol). In some embodiments, thebottom surface is functionalized with biotinlyated bovine serum albumin(BSA).

In certain embodiments, the bottom surface is functionalized with acoating comprising nitrodopa groups. In certain embodiments, the coatingcomprises groups of formula:

wherein R^(N) is an optionally substituted alkyl chain and

is hydrogen or a point of attachment to the surface. In someembodiments, R^(N) comprises a polymer. In some embodiments, R^(N)comprises a poly(lysine) or a poly(ethylene glycol). In someembodiments, R^(N) comprises a biotinylated poly(ethylene glycol). Insome embodiments, the coating comprises a co-polymer of poly(lysine)comprising lysine monomers, wherein the lysine monomers independentlycomprise PEG, biotinylated PEG, nitrodopa groups, phosphonate groups, orsilanes. In certain embodiments, the coating comprises a polymer offormula (P):

In some embodiments, X is —OMe, a biotin group, phosphonate, or silane.In some embodiments, each of i, j, k, and l is independently an integerbetween 0 and 100, inclusive.

In some embodiments, the bottom surface is functionalized with a silanecomprising an alkyl chain. In some embodiments, the bottom surface isfunctionalized with a silane comprising an optionally substituted alkylchain. In some embodiments, the bottom surface is functionalized with asilane comprising a poly(ethylene glycol) chain. In some embodiments,the bottom surface is functionalized with a silane comprising a couplinggroup. For example the coupling group may comprise chemical moieties,such as amine groups, carboxyl groups, hydroxyl groups, sulfhydrylgroups, metals, chelators, and the like. Alternatively, they may includespecific binding elements, such as biotin, avidin, streptavidin,neutravidin, lectins, SNAP-Tags™ or substrates therefore, associative orbinding peptides or proteins, antibodies or antibody fragments, nucleicacids or nucleic acid analogs, or the like. Additionally, oralternatively, the coupling group may be used to couple an additionalgroup that is used to couple or bind with the molecule of interest,which may, in some cases include both chemical functional groups andspecific binding elements. By way of example, a coupling group, e.g.,biotin, may be deposited upon a substrate surface and selectivelyactivated in a given area. An intermediate binding agent, e.g.,streptavidin, may then be coupled to the first coupling group. Themolecule of interest, which in this particular example would bebiotinylated, is then coupled to the streptavidin

In some embodiments, the bottom surface is functionalized with a silanecomprising biotin, or an analog thereof. In some embodiments, the bottomsurface is functionalized with a silane comprising a poly(ethylene)glycol chain, wherein the poly(ethylene glycol) chain comprises biotin.In certain embodiments, the bottom surface is functionalized with amixture of silanes, wherein at least one type of silane comprises biotinand at least one type of silane does not comprise biotin. In someembodiments, the mixture comprises about 10 fold less, about 25 foldless, about 50 fold less, about 100 fold less, about 250 fold less,about 500 fold less, or about 1000 fold less of the biotinylated silanethan the silane not comprising biotin.

FIG. 9 depicts two exemplary routes to generating a functionalized glassbottom surface. In the top route the glass surface is exposed to amixture of a PEG-silane and a biotinylated-PEG-silane in a ratio of250:1. In the bottom route, the glass surface is first exposed toisocyanato-propyl-triethoxysilane (IPTES). Reaction of the isocyanategroups with a mixture of a PEG-amine and biotinylated-PEG-amine formsurea bonds and yields a surface with PEG and biotinylated-PEG chainsattached to the surface via silane groups.

FIG. 7 depicts a non-limiting exemplary process for preparing the samplewell surface from the fabricated chip to initiation of a sequencingreaction. The sample well is depicted with a bottom surface (unshadedrectangle) and sidewalls (shaded vertical rectangles). The sidewalls maybe comprised of multiple layers (e.g., Al, Al₂O₃, Ti, TiO₂, TiN). Instep (a) the sidewalls are deposited with a barrier layer of Al₂O₃. TheAl₂O₃ barrier layer is then coated, in step (b), with a PEG phosphonategroups, for example, by treating the surface with one or morePEG-phosphonic acids. In step (c), the bottom surface is functionalized,for example, with a mixture of PEG-silane and biotinylated-PEG-silane.The ovals represent individual biotin groups which may provide sites foran attachment of a single molecule or complex, such as a polymerasecomplex. In step (d), a polymerase complex is attached to a biotin groupon the bottom surface. The polymerase may be attached by way of abinding agent, such as streptavidin, and a biotin tag on the polymerasecomplex. The polymerase complex may further comprise a template nucleicacid and primer (not shown). Step (e) depicts the initiation of asequencing reaction by exposure of the immobilized polymerase complex toluminescently labeled nucleotides.

The polymerase complex may be immobilized on the surface by exposing thecomplex to the functionalized surface in a binding mixture. In someembodiments, the binding mixture comprises one or more salts. In someembodiments, a salt comprises potassium acetate. In some embodiments, asalt comprises calcium chloride. In some embodiments, a salt is presentin a concentration of between about 1 mM and about 10 mM. In someembodiments, a salt is present in a concentration of between about 10 mMand about 50 mM. In some embodiments, a salt is present in aconcentration of between about 50 mM and about 100 mM. In someembodiments, a salt is present in a concentration of between about 100mM and about 250 mM. In some embodiments, the concentration of potassiumacetate is about 75 mM. In some embodiments, the concentration ofcalcium chloride is about 10 mM. In some embodiments, the bindingmixture comprises a reducing agent. In some embodiments, a reducingagent comprises dithiothreitol (DTT). In some embodiments, the reducingagent is present in a concentration of between about 1 mM and about 20mM. In some embodiments, the concentration of dithiothreitol is about 5mM. In some embodiments, the binding mixture comprises a buffer. In someembodiments, a buffer comprises MOPS. In some embodiments, a buffer ispresent in a concentration of between about 10 mM and about 100 mM. Insome embodiments, the concentration of MOPS is about 50 mM. In someembodiments, a buffer is present at a pH of between about 5.5 and about6.5. In some embodiments, a buffer is present at a pH of between about6.5. and about 7.5 In some embodiments, a buffer is present at a pH ofbetween about 7.5. and about 8.5 In some embodiments, the bindingmixture comprises deoxynucleotide triphosphates (dNTPs). In someembodiments, the deoxynucleotide triphosphates are present in aconcentration of between 250 nM and 10 μM. In some embodiments, theconcentration of dNTPs is about 2 μM. In some embodiments, the bindingmixture comprises a surfactant. In some embodiments, the surfactant is aTween surfactant (e.g., Tween 20). In some embodiments, the surfactantis present in a volume percent of between about 0.01% and about 0.1%. Insome embodiments, the volume percent of Tween is about 0.03%.

Polymerases

The term “polymerase,” as used herein, generally refers to any enzyme(or polymerizing enzyme) capable of catalyzing a polymerizationreaction. Examples of polymerases include, without limitation, a nucleicacid polymerase, a transcriptase or a ligase. A polymerase can be apolymerization enzyme.

Embodiments directed towards single molecule nucleic acid extension(e.g., for nucleic acid sequencing) may use any polymerase that iscapable of synthesizing a nucleic acid complementary to a target nucleicacid molecule. In some embodiments, a polymerase may be a DNApolymerase, an RNA polymerase, a reverse transcriptase, and/or a mutantor altered form of one or more thereof.

Examples of polymerases include, but are not limited to, a DNApolymerase, an RNA polymerase, a thermostable polymerase, a wild-typepolymerase, a modified polymerase, E. coli DNA polymerase I, T7 DNApolymerase, bacteriophage T4 DNA polymerase φ29 (psi29) DNA polymerase,Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, Pwopolymerase, Vent® polymerase, Deep Vent™ polymerase, Ex Taq™ polymerase,LA Taq™ polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mthpolymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tnepolymerase, Tma polymerase, Tca polymerase, Tih polymerase, Tfipolymerase, Platinum® Taq polymerases, Tbr polymerase, Tfl polymerase,Tth polymerase, Pfuturbo® polymerase, Pyrobest™ polymerase, Pwopolymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenowfragment, polymerase with 3′ to 5′ exonuclease activity, and variants,modified products and derivatives thereof. In some embodiments, thepolymerase is a single subunit polymerase. Non-limiting examples of DNApolymerases and their properties are described in detail in, among otherplaces, DNA Replication 2nd edition, Kornberg and Baker, W. H. Freeman,New York, N.Y. (1991).

Upon base pairing between a nucleobase of a target nucleic acid and thecomplementary dNTP, the polymerase incorporates the dNTP into the newlysynthesized nucleic acid strand by forming a phosphodiester bond betweenthe 3′ hydroxyl end of the newly synthesized strand and the alphaphosphate of the dNTP. In examples in which the luminescent tagconjugated to the dNTP is a fluorophore, its presence is signaled byexcitation and a pulse of emission is detected during or after the stepof incorporation. For detection labels that are conjugated to theterminal (gamma) phosphate of the dNTP, incorporation of the dNTP intothe newly synthesized strand results in release the beta and gammaphosphates and the detection label, which is free to diffuse in thesample well, resulting in a decrease in emission detected from thefluorophore.

In some embodiments, the polymerase is a polymerase with highprocessivity. However, in some embodiments, the polymerase is apolymerase with reduced processivity. Polymerase processivity generallyrefers to the capability of a polymerase to consecutively incorporatedNTPs into a nucleic acid template without releasing the nucleic acidtemplate.

In some embodiments, the polymerase is a polymerase with low 5′-3′exonuclease activity and/or 3′-5′ exonuclease. In some embodiments, thepolymerase is modified (e.g., by amino acid substitution) to havereduced 5′-3′ exonuclease activity and/or 3′-5′ activity relative to acorresponding wild-type polymerase. Further non-limiting examples of DNApolymerases include 9° Nm™ DNA polymerase (New England Biolabs), and aP680G mutant of the Klenow exo-polymerase (Tuske et al. (2000) JBC275(31):23759-23768). In some embodiments, a polymerase having reducedprocessivity provides increased accuracy for sequencing templatescontaining one or more stretches of nucleotide repeats (e.g., two ormore sequential bases of the same type).

In some embodiments, the polymerase is a polymerase that has a higheraffinity for a labeled nucleotide than for a non-labeled nucleic acid.

Embodiments directed toward single molecule RNA extension (e.g., for RNAsequencing) may use any reverse transcriptase that is capable ofsynthesizing complementary DNA (cDNA) from an RNA template. In suchembodiments, a reverse transcriptase can function in a manner similar topolymerase in that cDNA can be synthesized from an RNA template via theincorporation of dNTPs to a reverse transcription primer annealed to anRNA template. The cDNA can then participate in a sequencing reaction andits sequence determined as described above and elsewhere herein. Thedetermined sequence of the cDNA can then be used, via sequencecomplementarity, to determine the sequence of the original RNA template.Examples of reverse transcriptases include Moloney Murine Leukemia Virusreverse transcriptase (M-MLV), avian myeloblastosis virus (AMV) reversetranscriptase, human immunodeficiency virus reverse transcriptase(HIV-1) and telomerase reverse transcriptase.

The processivity, exonuclease activity, relative affinity for differenttypes of nucleic acid, or other property of a nucleic acid polymerasecan be increased or decreased by one of skill in the art by mutation orother modification relative to a corresponding wild-type polymerase.

Templates

The present disclosure provides devices, systems and methods fordetecting biomolecules or subunits thereof, such as nucleic acidmolecules. Such detection can include sequencing. A biomolecule may beextracted from a biological sample obtained from a subject (e.g., ahuman or other subject). In some embodiments, the subject may be apatient. In some embodiments, a target nucleic acid may be detectedand/or sequenced for diagnostic, prognostic, and/or therapeuticpurposes. In some embodiments, information for a sequencing assay may beuseful to assist in the diagnosis, prognosis, and/or treatment of adisease or condition. In some embodiments, the subject may be suspectedof having a health condition, such as a disease (e.g., cancer). In someembodiments, the subject may be undergoing treatment for a disease.

In some embodiments, a biological sample may be extracted from a bodilyfluid or tissue of a subject, such as breath, saliva, urine, blood(e.g., whole blood or plasma), stool, or other bodily fluid or biopsysample. In some examples, one or more nucleic acid molecules areextracted from the bodily fluid or tissue of the subject. The one ormore nucleic acids may be extracted from one or more cells obtained fromthe subject, such as part of a tissue of the subject, or obtained from acell-free bodily fluid of the subject, such as whole blood.

A biological sample may be processed in preparation for detection (e.g.,sequencing). Such processing can include isolation and/or purificationof the biomolecule (e.g., nucleic acid molecule) from the biologicalsample, and generation of more copies of the biomolecule. In someexamples, one or more nucleic acid molecules are isolated and purifiedfrom a bodily fluid or tissue of the subject, and amplified throughnucleic acid amplification, such as polymerase chain reaction (PCR).Then, the one or more nucleic acid molecules or subunits thereof can beidentified, such as through sequencing. However, in some embodimentsnucleic acid samples can be evaluated (e.g., sequenced) as described inthis application without requiring amplification.

As described in this application, sequencing can include thedetermination of individual subunits of a template biomolecule (e.g.,nucleic acid molecule) by synthesizing another biomolecule that iscomplementary or analogous to the template, such as by synthesizing anucleic acid molecule that is complementary to a template nucleic acidmolecule and identifying the incorporation of nucleotides with time(e.g., sequencing by synthesis). As an alternative, sequencing caninclude the direct identification of individual subunits of thebiomolecule.

During sequencing, signals indicative of individual subunits of abiomolecule may be collected in memory and processed in real time or ata later point in time to determine a sequence of the biomolecule. Suchprocessing can include a comparison of the signals to reference signalsthat enable the identification of the individual subunits, which in somecases yields reads. Reads may be sequences of sufficient length (e.g.,at least about 30, 50, 100 base pairs (bp) or more) that can be used toidentify a larger sequence or region, e.g., that can be aligned to alocation on a chromosome or genomic region or gene.

Sequence reads can be used to reconstruct a longer region of a genome ofa subject (e.g., by alignment). Reads can be used to reconstructchromosomal regions, whole chromosomes, or the whole genome. Sequencereads or a larger sequence generated from such reads can be used toanalyze a genome of a subject, such as to identify variants orpolymorphisms. Examples of variants include, but are not limited to,single nucleotide polymorphisms (SNPs) including tandem SNPs,small-scale multi-base deletions or insertions, also referred to asindels or deletion insertion polymorphisms (DIPs), Multi-NucleotidePolymorphisms (MNPs), Short Tandem Repeats (STRs), deletions, includingmicrodeletions, insertions, including microinsertions, structuralvariations, including duplications, inversions, translocations,multiplications, complex multi-site variants, copy number variations(CNV). Genomic sequences can comprise combinations of variants. Forexample, genomic sequences can encompass the combination of one or moreSNPs and one or more CNVs.

The term “genome” generally refers to an entirety of an organism'shereditary information. A genome can be encoded either in DNA or in RNA.A genome can comprise coding regions that code for proteins as well asnon-coding regions. A genome can include the sequence of all chromosomestogether in an organism. For example, the human genome has a total of 46chromosomes. The sequence of all of these together constitutes the humangenome. In some embodiments, the sequence of an entire genome isdetermined. However, in some embodiments, sequence information for asubset of a genome (e.g., one or a few chromosomes, or regions thereof)or for one or a few genes (or fragments thereof) is sufficient fordiagnostic, prognostic, and/or therapeutic applications.

Nucleic acid sequencing of a plurality of single-stranded target nucleicacid templates may be completed where multiple sample wells (e.g.,nanoapertures) are available, as is the case in devices describedelsewhere herein. Each sample well can be provided with asingle-stranded target nucleic acid template and a sequencing reactioncan be completed in each sample well. Each of the sample wells may becontacted with the appropriate reagents (e.g., dNTPs, sequencingprimers, polymerase, co-factors, appropriate buffers, etc.) necessaryfor nucleic acid synthesis during a primer extension reaction and thesequencing reaction can proceed in each sample well. In someembodiments, the multiple sample wells are contacted with allappropriate dNTPs simultaneously. In other embodiments, the multiplesample wells are contacted with each appropriate dNTP separately andeach washed in between contact with different dNTPs. Incorporated dNTPscan be detected in each sample well and a sequence determined for thesingle-stranded target nucleic acid in each sample well as is describedelsewhere herein.

While some embodiments may be directed to diagnostic testing bydetecting single molecules in a specimen, the inventors have alsorecognized that the single molecule detection capabilities of thepresent disclosure may be used to perform polypeptide (e.g., protein)sequencing or nucleic acid (e.g., DNA, RNA) sequencing of one or morenucleic acid segments of, for example, genes.

In some aspects, methods described herein can be performed using one ormore devices or apparatuses described in more detail below.

Overview of the Apparatus

The inventors have further recognized and appreciated that a compact,high-speed apparatus for performing detection and quantitation of singlemolecules or particles could reduce the cost of performing complexquantitative measurements of biological and/or chemical samples andrapidly advance the rate of biochemical technological discoveries.Moreover, a cost-effective device that is readily transportable couldtransform not only the way bioassays are performed in the developedworld, but provide people in developing regions, for the first time,access to essential diagnostic tests that could dramatically improvetheir health and well-being. For example, embodiments described hereinmay be used for diagnostic tests of blood, urine and/or saliva that maybe used by individuals in their home, or by a doctor in a remote clinicin a developing country.

A pixelated sensor device with a large number of pixels (e.g., hundreds,thousands, millions or more) allows for the detection of a plurality ofindividual molecules or particles in parallel. The molecules may be, byway of example and not limitation, proteins and/or DNA. Moreover, ahigh-speed device that can acquire data at more than one hundred framesper second allows for the detection and analysis of dynamic processes orchanges that occur over time within the sample being analyzed.

The inventors have recognized and appreciated that one hurdle preventingbioassay equipment from being made more compact was the need to filterthe excitation light from causing undesirable detection events at thesensor. Optical filters used to transmit the desired signal light (theluminescence) and sufficiently block the excitation light can be thick,bulky, expensive, and intolerant to variations in the incidence angle oflight, preventing miniaturization. The inventors, however, recognizedand appreciated that using a pulsed excitation source can reduce theneed for such filtering or, in some cases, remove the need for suchfilters altogether. By using sensors capable of determining the time aphoton is detected relative to the excitation light pulse, the signallight can be separated from the excitation light based on the time thatthe photon is received, rather than the spectrum of the light received.Accordingly, the need for a bulky optical filter is reduced and/orremoved in some embodiments.

The inventors have recognized and appreciated that luminescence lifetimemeasurements may also be used to identify the molecules present in asample. An optical sensor capable of detecting when a photon is detectedis capable of measuring, using the statistics gathered from many events,the luminescence lifetime of the molecule being excited by theexcitation light. In some embodiments, the luminescence lifetimemeasurement may be made in addition to a spectral measurement of theluminescence. Alternatively, a spectral measurement of the luminescencemay be completely omitted in identifying the sample molecule.Luminescence lifetime measurements may be made with a pulsed excitationsource. Additionally, luminescence lifetime measurements may be madeusing an integrated device that includes the sensor, or a device wherethe light source is located in a system separate from the integrateddevice.

The inventors have also recognized and appreciated that integrating asample well (e.g., a nanoaperture) and a sensor in a single integrateddevice capable of measuring luminescent light emitted from biologicalsamples reduces the cost of producing such a device such that disposablebioanalytical chips may be formed. Disposable, single-use integrateddevices that interface with a base instrument may be used anywhere inthe world, without the constraint of requiring high-cost biologicallaboratories for sample analyses. Thus, automated bioanalytics may bebrought to regions of the world that previously could not performquantitative analysis of biological samples. For example, blood testsfor infants may be performed by placing a blood sample on a disposableintegrated device, placing the disposable integrated device into asmall, portable base instrument for analysis, and processing the resultsby a computer for immediate review by a user. The data may also betransmitted over a data network to a remote location to be analyzed,and/or archived for subsequent clinical analyses.

The inventors have also recognized and appreciated that a disposable,single-use device may be made more simply and for lower cost by notincluding the light source on the chip. Instead, the light source may bereusable components incorporated into a system that interfaces with thedisposable chip to analyze a sample.

The inventors have also recognized and appreciated that, when a sampleis tagged with a plurality of different types of luminescent markers,any suitable characteristic of luminescent markers may be used toidentify the type of marker that is present in a particular pixel of thechip. For example, characteristics of the luminescence emitted by themarkers and/or characteristics of the excitation absorption may be usedto identify the markers. In some embodiments, the emission energy of theluminescence (which is directly related to the wavelength of the light)may be used to distinguish a first type of marker from a second type ofmarker. Additionally, or alternatively, luminescence lifetimemeasurements may also be used to identify the type of marker present ata particular pixel. In some embodiments, luminescence lifetimemeasurements may be made with a pulsed excitation source using a sensorcapable of distinguishing a time when a photon is detected withsufficient resolution to obtain lifetime information. Additionally, oralternatively, the energy of the excitation light absorbed by thedifferent types of markers may be used to identify the type of markerpresent at a particular pixel. For example, a first marker may absorblight of a first wavelength, but not equally absorb light of a secondwavelength, while a second marker may absorb light of the secondwavelength, but not equally absorb light of the first wavelength. Inthis way, when more than one excitation light source, each with adifferent excitation energy, may be used to illuminate the sample in aninterleaved manner, the absorption energy of the markers can be used toidentify which type of marker is present in a sample. Different markersmay also have different luminescent intensities. Accordingly, thedetected intensity of the luminescence may also be used to identify thetype of marker present at a particular pixel.

I. Overview of the System

The system includes an integrated device and an instrument configured tointerface with the integrated device. The integrated device includes anarray of pixels, where a pixel includes a sample well and at least onesensor. A surface of the integrated device has a plurality of samplewells, where a sample well is configured to receive a sample from aspecimen placed on the surface of the integrated device. A specimen maycontain multiple samples, and in some embodiments, different types ofsamples. The plurality of sample wells may have a suitable size andshape such that at least a portion of the sample wells receive onesample from a specimen. In some embodiments, the number of sampleswithin a sample well may be distributed among the sample wells such thatsome sample wells contain one sample with others contain zero, two ormore samples.

In some embodiments, a specimen may contain multiple single-stranded DNAtemplates, and individual sample wells on a surface of an integrateddevice may be sized and shaped to receive a single-stranded DNAtemplate. Single-stranded DNA templates may be distributed among thesample wells of the integrated device such that at least a portion ofthe sample wells of the integrated device contain a single-stranded DNAtemplate. The specimen may also contain tagged dNTPs which then enter inthe sample well and may allow for identification of a nucleotide as itis incorporated into a strand of DNA complementary to thesingle-stranded DNA template in the sample well. In such an example, the“sample” may refer to both the single-stranded DNA and the tagged dNTPcurrently being incorporated by a polymerase. In some embodiments, thespecimen may contain single-stranded DNA templates and tagged dNTPS maybe subsequently introduced to a sample well as nucleotides areincorporated into a complementary strand of DNA within the sample well.In this manner, timing of incorporation of nucleotides may be controlledby when tagged dNTPs are introduced to the sample wells of an integrateddevice.

Excitation energy is provided from an excitation source located separatefrom the pixel array of the integrated device. The excitation energy isdirected at least in part by elements of the integrated device towardsone or more pixels to illuminate an illumination region within thesample well. A marker or tag may then emit emission energy when locatedwithin the illumination region and in response to being illuminated byexcitation energy. In some embodiments, one or more excitation sourcesare part of the instrument of the system where components of theinstrument and the integrated device are configured to direct theexcitation energy towards one or more pixels.

Emission energy emitted by a sample may then be detected by one or moresensors within a pixel of the integrated device. Characteristics of thedetected emission energy may provide an indication for identifying themarked associated with the emission energy. Such characteristics mayinclude any suitable type of characteristic, including an arrival timeof photons detected by a sensor, an amount of photons accumulated overtime by a sensor, and/or a distribution of photons across two or moresensors. In some embodiments, a sensor may have a configuration thatallows for the detection of one or more timing characteristicsassociated with a sample's emission energy (e.g., fluorescencelifetime). The sensor may detect a distribution of photon arrival timesafter a pulse of excitation energy propagates through the integrateddevice, and the distribution of arrival times may provide an indicationof a timing characteristic of the sample's emission energy (e.g., aproxy for fluorescence lifetime). In some embodiments, the one or moresensors provide an indication of the probability of emission energyemitted by the marker or tag (e.g., fluorescence intensity). In someembodiments, a plurality of sensors may be sized and arranged to capturea spatial distribution of the emission energy. Output signals from theone or more sensors may then be used to distinguish a marker from amonga plurality of markers, where the plurality of markers may be used toidentify a sample within the specimen. In some embodiments, the In someembodiments, a sample may be excited by multiple excitation energies,and emission energy and/or timing characteristics of the emission energyemitted by the sample in response to the multiple excitation energiesmay distinguish a marker from a plurality of markers.

A schematic overview of the system 23-100 is illustrated in FIGS. 23Aand 23B. The system comprises both an integrated device 23-102 thatinterfaces with an instrument 23-104. In some embodiments, instrument23-104 may include one or more excitation sources 23-106 integrated aspart of instrument 23-104. In some embodiments, an excitation source maybe external to both instrument 23-104 and integrated device 23-102, andinstrument 23-104 may be configured to receive excitation energy fromthe excitation source and direct it to the integrated device. Theintegrated device may interface with the instrument using any suitablesocket for receiving the integrated device and holding it in preciseoptical alignment with the excitation source. The excitation source23-106 may be configured to provide excitation energy to the integrateddevice 23-102. As illustrated schematically in FIG. 23B, the integrateddevice 23-102 has multiple pixels, where at least a portion of pixels23-112 may perform independent analysis of a sample. Such pixels 23-112may be referred to as “passive source pixels” since a pixel receivesexcitation energy from a source 23-106 separate from the pixel, wherethe source excites a plurality of pixels. A pixel 23-112 has a samplewell 23-108 configured to receive a sample and a sensor 23-110 fordetecting emission energy emitted by the sample in response toilluminating the sample with excitation energy provided by theexcitation source 23-106. Sample well 23-108 may retain the sample inproximity to a surface of integrated device 23-102 to provide ease indelivery of excitation energy to the sample and detection of emissionenergy from the sample.

Optical elements for guiding and coupling excitation energy to thesample well 23-108 are located both on integrated device 23-102 and theinstrument 23-104. Such source-to-well elements may comprise one or moregrating couplers located on integrated device 23-102 to coupleexcitation energy to the integrated device and waveguides to deliverexcitation energy from instrument 23-104 to sample wells in pixels23-112. In some embodiments, elements located on the integrated devicemay act to direct emission energy from the sample well towards thesensor. Sample well 23-108, a portion of the excitation source-to-welloptics, and the sample well-to-sensor optics are located on integrateddevice 23-102. Excitation source 23-106 and a portion of thesource-to-well components are located in instrument 23-104. In someembodiments, a single component may play a role in both couplingexcitation energy to sample well 23-108 and delivering emission energyfrom sample well 23-108 to sensor 23-110. Examples of suitablecomponents, for coupling excitation energy to a sample well and/ordirecting emission energy to a sensor, to include in an integrateddevice are described in U.S. patent application Ser. No. 14/821,688entitled “INTEGRATED DEVICE FOR PROBING, DETECTING AND ANALYZINGMOLECULES,” and U.S. patent application Ser. No. 14/543,865 entitled“INTEGRATED DEVICE WITH EXTERNAL LIGHT SOURCE FOR PROBING, DETECTING,AND ANALYZING MOLECULES,” both of which are incorporated by reference intheir entirety.

As illustrated in FIG. 23B, the integrated device comprises a pluralityof pixels where a pixel 23-112 is associated with its own individualsample well 23-108 and at least one sensor 23-110. The plurality ofpixels may be arranged in an array, and there may be any suitable numberof pixels in the array. The number of pixels in integrated device 23-102may be in the range of approximately 10,000 pixels to 1,000,000 pixelsor any value or range of values within that range. In some embodiments,the pixels may be arranged in an array of 512 pixels by 512 pixels.Integrated device 23-102 and instrument 23-104 may includemulti-channel, high-speed communication links for handling dataassociated with large pixel arrays (e.g., more than 10,000 pixels).

Instrument 23-104 interfaces with integrated device 23-102 throughintegrated device interface 23-114. Integrated device interface 23-114may include components to position and/or align integrated device 23-102to instrument 23-104 to improve coupling of excitation energy fromexcitation source 23-106 to integrated device 23-102. Excitation source23-106 may be any suitable light source that is arranged to deliverexcitation energy to at least one sample well. Examples of suitableexcitation sources are described in U.S. patent application Ser. No.14/821,688 entitled “INTEGRATED DEVICE FOR PROBING, DETECTING ANDANALYZING MOLECULES,” which is incorporated by reference in itsentirety. In some embodiments, excitation source 23-106 includesmultiple excitation sources that are combined to deliver excitationenergy to integrated device 23-102. The multiple excitation sources maybe configured to produce multiple excitation energies or wavelengths.The integrated device interface 23-114 may receive readout signals fromthe sensors in the pixels located on the integrated device. Theintegrated device interface 23-114 may be designed such that theintegrated device attaches to the instrument by securing the integrateddevice to the integrated device interface 23-114.

The instrument 23-104 includes a user interface 23-116 for controllingthe operation of instrument 23-104. The user interface 23-116 isconfigured to allow a user to input information into the instrument,such as commands and/or settings used to control the functioning of theinstrument. In some embodiments, the user interface 23-116 may includebuttons, switches, dials, and a microphone for voice commands.Additionally, the user interface 23-116 may allow a user to receivefeedback on the performance of the instrument and/or integrated device,such as proper alignment and/or information obtained by readout signalsfrom the sensors on the integrated device. In some embodiments, the userinterface 23-116 may provide feedback using a speaker to provide audiblefeedback, and indicator lights and/or display screen for providingvisual feedback. In some embodiments, the instrument 23-104 includes acomputer interface 23-118 used to connect with a computing device23-120. Any suitable computer interface 23-118 and computing device23-120 may be used. For example, the computer interface 23-118 may be aUSB interface or a FireWire interface. The computing device 23-120 maybe any general purpose computer, such as a laptop or desktop computer.The computer interface 23-118 facilitates communication of informationbetween the instrument 23-104 and the computing device 23-120. Inputinformation for controlling and/or configuring the instrument 23-104 maybe provided through the computing device 23-120 connected to thecomputer interface 23-118 of the instrument. Output information may bereceived by the computing device 23-120 through the computer interface23-118. Such output information may include feedback about performanceof the instrument 23-104 and/or integrated device 23-112 and informationfrom the readout signals of the sensor 23-110. The instrument 23-104 mayalso include a processing device 23-122 for analyzing data received fromthe sensor 23-110 and/or sending control signals to the excitationsource 23-106. In some embodiments, the processing device 23-122 maycomprise a general purpose processor, a specially-adapted processor(e.g., a central processing unit (CPU) such as one or moremicroprocessor or microcontroller cores, a field-programmable gate array(FPGA), an application-specific integrated circuit (ASIC), a customintegrated circuit, a digital signal processor (DSP), or a combinationthereof.) In some embodiments, the processing of data from the sensor23-110 may be performed by both the processing device 23-122 and theexternal computing device 23-120. In other embodiments, the computingdevice 23-120 may be omitted and processing of data from the sensor23-110 may be performed solely by processing device 23-122.

A cross-sectional schematic of the integrated device 24-102 illustratinga row of pixels is shown in FIG. 24A. Each pixel 24-112 includes asample well 24-108 and a sensor 24-110. The sensor 24-110 may be alignedand positioned to sample well 24-112 such that sensor 24-110 receivesemission energy emitted by a sample within sample well 24-112. Examplesof suitable sensors are described in U.S. patent application Ser. No.14/821,656 entitled “INTEGRATED DEVICE FOR TEMPORAL BINNING OF RECEIVEDPHOTONS,” which is incorporated by reference in its entirety.

An excitation source coupled to the integrated device may provideexcitation energy to one or more pixels of integrated device 24-102.FIG. 24B is a schematic illustrating coupling of excitation source24-106 to integrated device 24-102 to provide excitation energy 24-130(shown in dashed lines) to integrated device 24-102. FIG. 24Billustrates the path of excitation energy from excitation energy source24-106 to a sample well 24-108 in pixel 24-112. Components located offof the integrated device may be used to position and align theexcitation source 24-106 to the integrated device. Such components mayinclude optical components including lenses, mirrors, prisms, apertures,attenuators, and/or optical fibers. Additional mechanical components maybe included in the instrument to allow for control of one or morealignment components. Such mechanical components may include actuators,stepper motors, and/or knobs.

The integrated device includes components that direct the excitationenergy 24-130 towards pixels in the integrated device. Within each pixel24-112, excitation energy is coupled to the sample well 24-108associated with the pixel. Although FIG. 24B illustrates excitationenergy coupling to each sample well in a row of pixels, in someembodiments, excitation energy may not couple to all of the pixels in arow. In some embodiments, excitation energy may couple to a portion ofpixels or sample wells in a row of pixels of the integrated device.Excitation energy may illuminate a sample located within a sample well.The sample may reach an excited state in response to being illuminatedby the excitation energy. When a sample is in an excited state, thesample may emit emission energy and the emission energy may be detectedby a sensor. FIG. 24B schematically illustrates the path of emissionenergy 24-140 (shown as solid lines) from sample well 24-108 to sensor24-110 of pixel 24-112. Sensor 24-110 in pixel 24-112 may be configuredand positioned to detect emission energy from sample well 24-108. Insome embodiments, sensor 24-110 may include multiple sub-sensors.

A sample to be analyzed may be introduced into sample well 24-108 ofpixel 24-112. The sample may be a biological sample or any othersuitable sample, such as a chemical sample. The sample may includemultiple molecules and the sample well may be configured to isolate asingle molecule. In some instances, the dimensions of the sample wellmay act to confine a single molecule within the sample well, allowingmeasurements to be performed on the single molecule. An excitationsource 24-106 may be configured to deliver excitation energy into thesample well 24-108, so as to excite the sample or at least oneluminescent marker attached to the sample or otherwise associated withthe sample while it is within an illumination area within the samplewell 24-108.

When an excitation source delivers excitation energy to a sample well,at least one sample within the well may luminesce, and the resultingemission may be detected by a sensor. As used herein, the phrases “asample may luminesce” or “a sample may emit radiation” or “emission froma sample” mean that a luminescent tag, marker, or reporter, the sampleitself, or a reaction product associated with the sample may produce theemitted radiation.

One or more components of an integrated device may direct emissionenergy towards a sensor. The emission energy or energies may be detectedby the sensor and converted to at least one electrical signal. Theelectrical signals may be transmitted along conducting lines in thecircuitry of the integrated device connected to the instrument throughthe integrated device interface, such as integrated device interface23-114 of instrument 23-104 shown in FIG. 23B. The electrical signalsmay be subsequently processed and/or analyzed. Processing or analyzingof electrical signals may occur on a suitable computing device eitherlocated on the instrument 23-104 or off instrument, such as computingdevice 23-120 shown in FIG. 23B.

In operation, parallel analyses of samples within the sample wells arecarried out by exciting the samples within the wells using theexcitation source and detecting signals from sample emission with thesensors. Emission energy from a sample may be detected by acorresponding sensor and converted to at least one electrical signal.The resulting signal, or signals, may be processed on the integrateddevice in some embodiments, or transmitted to the instrument forprocessing by the processing device and/or computing device. Signalsfrom a sample well may be received and processed independently fromsignals associated with the other pixels.

In some embodiments, a sample may be labeled with one or more markers,and emission associated with the markers is discernable by theinstrument. For example the sensor may be configured to convert photonsfrom the emission energy into electrons to form an electrical signalthat may be used to discern a lifetime that is dependent on the emissionenergy from a specific marker. By using markers with different lifetimesto label samples, specific samples may be identified based on theresulting electrical signal detected by the sensor.

A sample may contain multiple types of molecules and differentluminescent markers may uniquely associate with a molecule type. Duringor after excitation, the luminescent marker may emit emission energy.One or more properties of the emission energy may be used to identifyone or more types of molecules in the sample. Properties of the emissionenergy used to distinguish among types of molecules may include afluorescence lifetime value, intensity, and/or emission wavelength. Asensor may detect photons, including photons of emission energy, andprovide electrical signals indicative of one or more of theseproperties. In some embodiments, electrical signals from a sensor mayprovide information about a distribution of photon arrival times acrossone or more time intervals. The distribution of photon arrival times maycorrespond to when a photon is detected after a pulse of excitationenergy is emitted by an excitation source. A value for a time intervalmay correspond to a number of photons detected during the time interval.Relative values across multiple time intervals may provide an indicationof a temporal characteristic of the emission energy (e.g., lifetime).Analyzing a sample may include distinguishing among markers by comparingvalues for two or more different time intervals within a distribution.In some embodiments, an indication of the intensity may be provided bydetermining a number of photons across all time bins in a distribution.

II. Integrated Device

The integrated device may be configured to receive excitation energyfrom an external excitation energy source. In some embodiments, a regionof the device may be used to couple to an excitation energy sourcelocated off the integrated device. Components of the integrated devicemay guide excitation energy from the excitation source coupling regionto at least one pixel. In some embodiments, at least one waveguide maybe configured to deliver excitation energy to at least one pixel havinga sample well. A sample located within the sample well may emit emissionenergy in response to being illuminated with excitation energy. One ormore sensors located within the pixel are configured to receive theemission energy.

Components and/or layers of integrated device 25-200 according to someembodiments shown in FIG. 25 include a sample well 25-203, waveguide25-220, and sensor 25-275 integrated into one device. Sample well 25-203may be formed in sample well layer 25-201 of integrated device 25-200.In some embodiments, the sample well layer 25-201 may be metal. Samplewell 25-203 may have a dimension D_(tv) which may indicate across-sectional dimension of the sample well. Sample well 25-203 may actas a nanoaperture and have one or more sub-wavelength dimensions thatcreate a field enhancement effect that increases the intensity of theexcitation of the sample in sample well 25-203. Waveguide 25-220 isconfigured to deliver excitation energy from excitation source 25-230located off device 25-200 to sample well 25-203. The waveguide 25-220may be formed in a layer between sample well layer 25-201 and sensor25-275. The design of integrated device 25-200 allows for sensor 25-275to collect luminescence emitted from a sample in sample well 25-203. Atleast some of the time, the sample absorbs excitation energy and emits aphoton with an energy less than that of the excitation energy, referredto as emission energy or luminescence.

Having sample well 25-203 and sensor 25-275 on integrated device 25-200may reduce the optical distance that light travels from the sample well25-203 to sensor 25-215. Dimensions of integrated device 25-200 orcomponents within the device may be configured for a certain opticaldistance. Optical properties of the materials of components and/or oneor more layers of the device may determine an optical distance between asample well and a sensor. In some embodiments, the thicknesses of one ormore layers may determine the optical distance between the sample welland sensor in a pixel. Additionally or alternatively, the index ofrefraction of materials that form one or more layers of integrateddevice 25-200 may determine the optical distance between sample well25-203 and sensor 25-275 in a pixel. Such an optical distance betweenthe sample well and sensor in a pixel may be less than 1 mm, less than100 microns, less than 25 microns, and/or less than 10 microns. One ormore layers may be present between sample well layer 25-201 andwaveguide layer 25-220 to improve coupling of excitation energy fromwaveguide 25-220 to sample well 25-203. Although integrated device25-200 shown in FIG. 25 illustrates only a single layer 25-210, multiplelayers may be formed between sample well 25-203 and waveguide 25-220.Layer 25-210 may be formed with optical properties to improve couplingof excitation energy from waveguide 25-220 to sample well 25-203. Layer25-210 may be configured to reduce scattering and/or absorption ofexcitation energy and/or increase luminescence from a sample in samplewell 25-203. Layer 25-210 may be optically transparent, according tosome embodiments, so that light may travel to and from the sample well25-203 with little attenuation. In some embodiments, dielectricmaterials may be used to form layer 25-210. In some embodiments,excitation energy coupling components within layer 25-210 and/or at theinterface between layer 25-210 and sample well layer 25-201 may beprovided to improve coupling of excitation energy from waveguide 25-220to sample well 25-203. As an example, energy-collection components25-215 formed at the interface between sample well layer 25-201 andlayer 25-210 may be configured to improve coupling of excitation energyfrom waveguide 25-220 to sample well 25-203. Energy-collectioncomponents 25-215 are optional and, in some embodiments, theconfiguration of waveguide 25-220 and sample well 25-203 may allow foradequate coupling excitation energy without the presence of excitationenergy collection components 25-215.

Luminescent light or energy emitted from a sample in the sample well25-203 may be transmitted to the sensor 25-275 in a variety of ways,some examples of which are described in detail below. Some embodimentsmay use optical components to increase the likelihood that light of aparticular wavelength is directed to an area or portion of the sensor25-275 that is dedicated to detecting light of that particularwavelength. The sensor 25-275 may include multiple portions fordetecting simultaneously light of different wavelengths that maycorrespond to emissions from different luminescent markers.

One or more layers may be present between sample well 25-203 and sensor25-275 that may be configured to improve collection of luminescence fromsample well 25-203 to sensor 25-275. Luminescence directing componentsmay be located at the interface between sample well layer 25-201 andlayer 25-210. The energy-collection components 25-215 may focus emissionenergy toward the sensor 25-275, and may additionally or alternativelyspatially separate emission energies that have different characteristicenergies or wavelengths. Such energy-collection components 25-215 mayinclude a grating structure for directing luminescence towards sensor25-275. In some embodiments, the grating structure may be a series ofconcentric rings or “bullseye” grating structure configuration. Theconcentric circular gratings may protrude from a bottom surface of thesample well layer 25-201. The circular gratings may act as plasmonicelements which may be used to decrease the spread of the signal lightand direct the signal light towards associated sensor 25-275. Such abullseye grating may direct luminescence more efficiently towards sensor25-275.

Layer 25-225 may be formed adjacent to the waveguide. The opticalproperties of layer 25-225 may be selected to improve collection ofluminescence from the sample well to sensor 25-275. In some embodiments,layer 25-225 may be a dielectric material. A baffle may be formedbetween sample well layer 25-201 and sensor 25-275. Baffle 25-240 may beconfigured such that the sensor 25-275 receives luminescencecorresponding to sample well 25-203 and reduces luminescence, andreflected/scattered excitation from other sample wells. Filteringelements 25-260 may be positioned and configured to reduce excitationenergy from reaching sensor 25-275. In some embodiments, filteringelements 25-260 may include a filter that selectively transmits emissionenergy of one or more markers used to label a sample. In embodimentswith an array of sample wells and an array of sensors where each samplewell has a corresponding sensor, a baffle corresponding to each samplewell may be formed to reduce luminescence from other sample wells andreflected and/or scattered excitation light from being collected by asensor corresponding to the sample well.

One or more layers may be formed between waveguide 25-220 and sensor25-275 to reduce transmission of excitation energy to sensor. In someembodiments, filtering elements may be formed between waveguide 25-220and sensor 25-275. Such filtering elements may be configured to reducetransmission of excitation energy to sensor 25-275 while allowingluminescence from the sample well to be collected by sensor 25-275.

The emission energy or energies may be detected by the sensor 25-275 andconverted to at least one electrical signal. The electrical signal orsignals may be transmitted along one or more row or column conductinglines (not shown) to the integrated electronic circuitry on thesubstrate 25-200 for subsequent signal processing.

The above description of FIG. 25 is an overview of some of thecomponents of the apparatus according to some embodiments. In someembodiments, one or more elements of FIG. 25 may be absent or in adifferent location. The components of the integrated device 25-200 andexcitation source 25-230 are described in more detail below.

A. Excitation Source Coupling Region

The integrated device may have an excitation source coupling regionconfigured to couple with an external excitation energy source and guideexcitation towards at least one pixel in a pixel area of the integrateddevice. The excitation source coupling region may include one or morestructures configured to couple light into at least one waveguide. Anysuitable mechanism for coupling excitation energy into a waveguide maybe used.

The excitation source coupling region of an integrated device mayinclude structural components configured to couple with an externalexcitation source. An integrated device may include a grating couplerconfigured to couple with an external excitation source positionedproximate a surface of the integrated device and direct light towards atleast one waveguide of the integrated device. Features of the gratingcoupler, such as the size, shape, and/or grating configurations may beformed to improve coupling of the excitation energy from the excitationsource to the waveguide. The grating coupler may include one or morestructural components where the spacing between the structuralcomponents may act to propagate light. One or more dimensions of thegrating coupler may provide desirable coupling of light having a certaincharacteristic wavelength.

An integrated device may also include a waveguide having a taperedregion at an end of the waveguide. One or more dimensions of thewaveguide perpendicular to a direction of light propagation in thewaveguide may be larger at an end of the waveguide, forming a taperedregion of the waveguide. In some embodiments, the tapered region of awaveguide may have a dimension perpendicular to the propagation of lightand parallel to a surface of the integrated device that is larger at anend of the waveguide and becomes smaller along the length of thewaveguide. In embodiments that include a grating coupler, the taperedregion can be positioned proximate to the grating coupler such that thelarger end of the tapered region is located closest to the gratingcoupler. The tapered region may be sized and shaped to improve couplingof light between the grating coupler and the waveguide by expanding oneor more dimensions of the waveguide to allow for improved mode overlapof the waveguide with the grating coupler. In this manner, an excitationsource positioned proximate the surface of an integrated device maycouple light into the waveguide via the grating coupler. Such acombination of grating coupler and waveguide taper may allow for moretolerance in the alignment and positioning of the excitation source tothe integrated device.

A grating coupler may be positioned in a region of the integrated deviceexternal to the pixels of the integrated device. On a surface of theintegrated device, the sample wells of the pixels may occupy a region ofthe surface separate from the excitation source coupling region. Anexcitation source positioned proximate to the surface of the excitationsource coupling region may couple with the grating coupler. The samplewells may be positioned separate from the excitation source couplingregion to reduce interference of light from the excitation source onperformance of the pixels. A grating coupler of an integrated device maybe formed within one or more layers of the integrated device thatinclude a waveguide. In this manner, an excitation source couplingregion of an integrated device may include a grating coupler within thesame plane of the integrated device as a waveguide. The grating couplermay be configured for a particular set of beam parameters, includingbeam width, angle of incidence, and/or polarization of the incidentexcitation energy.

A cross-sectional view of integrated device 26-200 is shown in FIG. 26.Integrated device 26-200 includes at least one sample well 26-222 formedin layer 26-223 of integrated device 26-200. Integrated device 26-200includes grating coupler 26-216 and waveguide 26-220 formed insubstantially the same plane of integrated device 26-200. In someembodiments, grating coupler 26-216 and waveguide 26-220 are formed fromthe same layer of integrated device 26-200 and may include the samematerial. Excitation source coupling region 26-201 of integrated device26-200 includes grating coupler 26-216. As shown in FIG. 26, sample well26-222 is positioned on a surface of integrated device 26-200 externalto excitation source coupling region 26-201. Excitation source 26-214positioned relative to integrated device 26-200 may provide excitationenergy incident on surface 26-215 of integrated device 26-200 withinexcitation source coupling region 26-201. By positioning grating coupler26-216 within excitation source coupling region 26-201, grating coupler26-216 may couple with the excitation energy from excitation source26-214 and couple excitation energy to waveguide 26-220. Waveguide26-220 is configured to propagate excitation energy to the proximity ofone or more sample wells 26-222.

A grating coupler may be formed from one or more materials. In someembodiments, a grating coupler may include alternating regions ofdifferent materials along a direction parallel to propagation of lightin the waveguide. As shown in FIG. 26, grating coupler 26-216 includesstructures that are surrounded by material 26-224. The one or morematerials that form a grating coupler may have one or more indices ofrefraction suitable for coupling and propagating light. In someembodiments, a grating coupler may include structures formed from onematerial surrounded by a material having a larger index of refraction.As an example, a grating coupler may include structures formed ofsilicon nitride and surrounded by silicon dioxide. Any suitabledimensions and/or inter-grating spacing may be used to form a gratingcoupler. Grating coupler 26-216 may have a dimension perpendicular tothe propagation of light through the waveguide, such as along they-direction as shown in FIG. 26, of approximately 50 nm, approximately100 nm, approximately 150 nm, or approximately 200 nm. Spacing betweenstructures of the grating coupler along a direction parallel to lightpropagation in the waveguide, such as along the z-direction as shown inFIG. 26, may have any suitable distance. The inter-spacing grating maybe approximately 300 nm, approximately, 350 nm, approximately, 400 nm,approximately 420 nm, approximately 450 nm, or approximately 500 nm. Insome embodiments, the inter-grating spacing may be variable within agrating coupler. Grating coupler 26-216 may have one or more dimensionssubstantially parallel to surface 26-215 of integrated device 26-200that provide a suitable area for coupling with external excitationsource 26-214. The area of grating coupler 26-216 may coincide with oneor more dimensions of a beam of light from excitation source 26-214 suchthat the beam overlaps with grating coupler 26-215. A grating couplermay have an area configured for a beam diameter of approximately 10microns, approximately 20 microns, approximately 30 microns, orapproximately 40 microns.

An integrated device may include a layer formed on a side of a gratingcoupler opposite to the excitation source configured reflect light. Thelayer may reflect excitation energy that passes through the gratingcoupler towards the grating coupler. By including the layer in anintegrated device, coupling efficiency of the excitation energy to thewaveguide may be improved. An example of a reflective layer is layer26-218 of integrated device 26-200 shown in FIG. 26. Layer 26-218 ispositioned within excitation source coupling region 26-201 of integrateddevice 26-200 and is configured to reflect light towards grating coupler26-216. Layer 26-218 is formed proximate to the side of grating coupler26-216 opposite to the incident excitation energy from excitation source26-214. Positioning layer 26-218 external to pixels of integrated device26-200 may reduce interference of layer 26-218 on the performancecapabilities of the pixels. Layer may 26-218 may include any suitablematerial. Layer 26-218 may be substantially reflective for one or moreexcitation energies. In some embodiments, this layer may include Al,AlCu, and/or TiN.

B. Waveguide

An integrated device may include one or more waveguides arranged todeliver a desired amount of excitation energy to one or more samplewells of the integrated device. A waveguide positioned in the vicinityof one or more sample wells such that as excitation energy propagatesalong the waveguide a portion of excitation energy couples to the one ormore sample wells. A waveguide may couple excitation energy to aplurality of pixels and act as a bus waveguide. For example, a singlewaveguide may deliver excitation energy to a row or a column of pixelsof an integrated device. In some embodiments, a waveguide may beconfigured to propagate excitation energy having a plurality ofcharacteristic wavelengths. A pixel of an integrated device may includeadditional structures (e.g., microcavity) configured to directexcitation energy from the waveguide toward the vicinity of the samplewell. In some embodiments, a waveguide may carry an optical mode havingan evanescent tail configured to extend into a sample well and/or in aregion in the vicinity of the sample well. Additional energy-couplingstructures located near the sample well may couple energy from theevanescent tail into the sample well.

One or more dimensions of a waveguide of an integrated device mayprovide desired propagation of excitation energy along the waveguideand/or into one or more sample wells. A waveguide may have a dimensionperpendicular to the propagation of light and parallel to a plane of thewaveguide, which may be considered as a cross-sectional width. Awaveguide may have a cross-sectional width of approximately 0.4 microns,approximately 0.5 microns, approximately 0.6 microns, approximately 0.65microns, approximately 0.8 microns, approximately 1 micron, orapproximately 1.2 microns. A waveguide may have a dimensionperpendicular to the propagation of light and perpendicular to a planeof the waveguide, which may be considered as a cross-sectional height. Awaveguide may have a cross-sectional height of approximately 0.05microns, approximately 0.1 microns, approximately 0.15 microns,approximately 0.16 microns, approximately 0.17 microns, approximately0.2 microns, or approximately 0.3 microns. In some embodiments, awaveguide has a larger cross-sectional width than cross-sectionalheight. A waveguide may be positioned a certain distance from one ormore sample wells within in integrated device, such as distance D shownin FIG. 26 between sample well 26-222 and waveguide 26-220, that isapproximately 0.3 microns, 0.5 microns, or approximately 0.7 microns.

In an exemplary embodiment, a waveguide may have a cross-sectional widthof approximately 0.5 μm and a cross-sectional height of approximately0.1 μm, and be positioned approximately 0.5 μm below the sample welllayer. In another exemplary embodiment, a waveguide may have across-sectional width of approximately 1 μm and a cross-sectional heightof 0.18 μm, and be positioned 0.3 μm below the sample well layer.

A waveguide may be dimensioned to support a single transverse radiationmode or may be dimensioned to support multi-transverse radiation modes.In some embodiments, one or more dimensions of a waveguide may act suchthat the waveguide sustains only a single transverse mode and mayselectively propagate TE or TM polarization modes. In someimplementations, a waveguide may have highly reflective sections formedon its ends, so that it supports a longitudinal standing mode within thewaveguide. By supporting one mode, the waveguide may have reduced modalinterference effects from cross coupling of modes having differentpropagation constants. In some embodiments, the highly reflectivesections comprise a single, highly reflective surface. In otherembodiments, the highly reflective sections comprise multiple reflectivestructures that, in aggregate, result in a high reflectance. Waveguidesmay be configured to split excitation energy from a single excitationsource having a higher output intensity using waveguide beam splittersto create a plurality of excitation energy beams from a singleexcitation source. Such beam splitters may include evanescent couplingmechanisms. Additionally or alternatively, photonic crystals may be usedin the waveguide structure to improve propagation of excitation energyand/or in the material surrounding the waveguide to reduce scattering ofexcitation energy.

The position and arrangement of the waveguide with respect to othercomponents in a pixel of the integrated devices may be configured toimprove coupling of excitation energy towards a sample well, improvecollection of emission energy by the sensor, and/or reduce signal noiseintroduced by excitation energy. A waveguide may be sized and/orpositioned relative to a sample well so as to reduce interference ofexcitation energy propagating in the waveguide with emission energyemitted from the sample well. Positioning and arrangement of a waveguidein an integrated device may depend on the refractive indices of thewaveguide and material surrounding the waveguide. For example, adimension of the waveguide perpendicular to a direction of lightpropagation along the waveguide and within a plane of the waveguide maybe increased so that a substantial amount of emission energy from asample well passes through the waveguide as it propagates to the sensorof the pixel. In some implementations, a distance between a sample welland waveguide and/or waveguide thickness may be selected to reducereflections from one or more interfaces between the waveguide and asurrounding material. According to some embodiments, reflection ofemission energy by a waveguide may be reduced to less than about 5% insome embodiments, less than about 2% in some embodiments, and yet lessthan about 1% in some embodiments.

The capability of a waveguide to propagate excitation energy may dependboth on the material for the waveguide and material surrounding thewaveguide. In this manner, the waveguide structure may include a corematerial, such as waveguide 26-220, and a cladding material, such asregion 26-224 as illustrated in FIG. 26. The material of both thewaveguide and surrounding material may allow for propagation ofexcitation energy having a characteristic wavelength through thewaveguide. Material for either the waveguide or the surrounding materialmay be selected for particular indices of refraction or combination ofindices of refraction. The waveguide material may have a lowerrefractive index than the waveguide surrounding material. Examplewaveguide materials include silicon nitride (Si_(x)N_(y)), siliconoxynitride, silicon carbide, tantalum oxide (TaO₂), aluminum dioxide.Example waveguide surrounding materials include silicon dioxide (SiO₂)and silicon oxide. The waveguide and/or the surrounding material mayinclude one or more materials. In some instances, a desired refractiveindex for a waveguide and/or surrounding material can be obtained byforming the waveguide and/or surrounding material to include more thanone material. In some embodiments, a waveguide comprises silicon nitrideand a surrounding material comprises silicon dioxide.

Splitting waveguides from a single waveguide to multiple waveguides mayallow for excitation energy to reach multiple rows or columns of samplewells of an integrated device. An excitation source may be coupled to aninput waveguide and the input waveguide may be split into multipleoutput waveguides, where each output waveguide delivers excitationenergy to a row or column of sample wells. Any suitable techniques forsplitting and/or combining waveguides may be used. Such waveguidesplitting and/or combining techniques may include a star splitter orcoupler, a Y splitter, and/or an evanescent coupler. Additionally oralternatively, a multi-mode interference splitter (MMI) may be used forsplitting and/or combining waveguides. One or more of these waveguidesplitting and/or combining techniques may be used for directingexcitation energy to a sample well.

C. Sample Well

According to some embodiments, a sample well 27-210 may be formed at oneor more pixels of an integrated device. A sample well may comprise asmall volume or region formed at a surface of a substrate 27-105 andarranged such that samples 27-101 may diffuse into and out of the samplewell from a specimen deposited on the surface of the substrate, asdepicted in FIG. 27A and FIG. 27B, which illustrate a single pixel27-100 of an integrated device. In various embodiments, a sample well27-210 may be arranged to receive excitation energy from a waveguide27-240. Samples 27-101 that diffuse into the sample well may beretained, temporarily or permanently, within an excitation region 27-215of the sample well by an adherent 27-211. In the excitation region, asample may be excited by excitation energy (e.g., excitation radiation27-247), and subsequently emit radiation that may be observed andevaluated to characterize the sample.

In further detail of operation, at least one sample 27-101 to beanalyzed may be introduced into a sample well 27-210, e.g., from aspecimen (not shown) containing a fluid suspension of samples.Excitation energy from a waveguide 27-240 may excite the sample or atleast one marker attached to the sample or included in a tag associatedwith the sample while it is within an excitation region 27-215 withinthe sample well. According to some embodiments, a marker may be aluminescent molecule (e.g., fluorophore) or quantum dot. In someimplementations, there may be more than one marker that is used toanalyze a sample (e.g., distinct markers and tags that are used forsingle-molecule genetic sequencing as described in “Real-Time DNASequencing from Single Polymerase Molecules,” by J. Eid, et al., Science323, p. 133 (2009), which is incorporated by reference in its entirety).During and/or after excitation, the sample or marker may emit emissionenergy. When multiple markers are used, they may emit at differentcharacteristic energies and/or emit with different temporalcharacteristics including different lifetimes. The emission energy fromthe sample well may radiate or otherwise travel to a sensor 27-260 wherethe emission energy is detected and converted into electrical signalsthat can be used to characterize the sample.

According to some embodiments, a sample well 27-210 may be a partiallyenclosed structure, as depicted in FIG. 27B. In some implementations, asample well 27-210 comprises a sub-micron-sized hole or opening(characterized by at least one transverse dimension D_(sw)) formed in atleast one layer of material 27-230. In some cases, the hole may bereferred to as a “nanoaperture.” The transverse dimension of the samplewell may be between approximately 20 nanometers and approximately 1micron, according to some embodiments, though larger and smaller sizesmay be used in some implementations. A volume of the sample well 27-210may be between about 10⁻²¹ liters and about 10⁻¹⁵ liters, in someimplementations. A sample well may be formed as a waveguide that may, ormay not, support a propagating mode. A sample well may be formed as awaveguide that may, or may not, support a propagating mode. In someembodiments, a sample well may be formed as a zero-mode waveguide (ZMW)having a cylindrical shape (or similar shape) with a diameter (orlargest transverse dimension) D_(sw). A ZMW may be formed in a singlemetal layer as a nano-scale hole that does not support a propagatingoptical mode through the hole.

Because the sample well 27-210 has a small volume, detection ofsingle-sample events (e.g., single-molecule events) at each pixel may bepossible even though samples may be concentrated in an examined specimenat concentrations that are similar to those found in naturalenvironments. For example, micromolar concentrations of the sample maybe present in a specimen that is placed in contact with the integrateddevice, but at the pixel level only about one sample (or single moleculeevent) may be within a sample well at any given time. Statistically,some sample wells may contain no samples and some may contain more thanone sample. However, an appreciable number of sample wells may contain asingle sample (e.g., at least 30% in some embodiments), so thatsingle-molecule analysis can be carried out in parallel for a largenumber of pixels. Because single-molecule or single-sample events may beanalyzed at each pixel, the integrated device makes it possible todetect rare events that may otherwise go unnoticed in ensemble averages.

A transverse dimension D_(sw) of a sample well may be between about 500nanometers (nm) and about one micron in some embodiments, between about250 nm and about 500 nm in some embodiments, between about 100 nm andabout 250 nm in some embodiments, and yet between about 20 nm and about100 nm in some embodiments. According to some implementations, atransverse dimension of a sample well is between approximately 80 nm andapproximately 180 nm, or between approximately one-quarter andone-eighth of the excitation wavelength or emission wavelength.According to other implementations, a transverse dimension of a samplewell is between approximately 120 nm and approximately 170 nm. In someembodiments, the depth or height of the sample well 27-210 may bebetween about 50 nm and about 500 nm. In some implementations, the depthor height of the sample well 27-210 may be between about 80 nm and about250 nm.

A sample well 27-210 having a sub-wavelength, transverse dimension canimprove operation of a pixel 27-100 of an integrated device in at leasttwo ways. For example, excitation energy incident on the sample wellfrom a side opposite the specimen may couple into the excitation region27-215 with an exponentially decreasing power, and not propagate throughthe sample well to the specimen. As a result, excitation energy isincreased in the excitation region where it excites a sample ofinterest, and is reduced in the specimen where it would excite othersamples that would contribute to background noise. Also, emission from asample retained at a base of the well (e.g., nearer to the sensor27-260) is preferably directed toward the sensor, since emissionpropagating up through the sample well is highly suppressed. Both ofthese effects can improve signal-to-noise ratio at the pixel. Theinventors have recognized several aspects of the sample well that can beimproved to further boost signal-to-noise levels at the pixel. Theseaspects relate to sample well shape and structure, and also to adjacentoptical and plasmonic structures (described below) that aid in couplingexcitation energy to the sample well and emitted radiation from thesample well.

According to some embodiments, a sample well 27-210 may be formed as ananoaperture configured to not support a propagating mode for particularwavelengths of interest. In some instances, the nanoaperture isconfigured where all modes are below a threshold wavelength and theaperture may be a sub-cutoff nanoaperture (SCN). For example, the samplewell 27-210 may comprise a cylindrically-shaped hole or bore in aconductive layer. The cross-section of a sample well need not be round,and may be elliptical, square, rectangular, or polygonal in someembodiments. Excitation energy 27-247 (e.g., visible or near infraredradiation) may enter the sample well through an entrance aperture 27-212that may be defined by walls 27-214 of the sample well at a first end ofthe well, as depicted in FIG. 27B. When formed as a SCN, the excitationenergy may decay exponentially along a length of the nanoaperture (e.g.in the direction of the specimen). In some implementations, thewaveguide may comprise a SCN for emitted radiation from the sample, butmay not be a SCN for excitation energy. For example, the aperture andwaveguide formed by the sample well may be large enough to support apropagating mode for the excitation energy, since it may have a shorterwavelength than the emitted radiation. The emission, at a longerwavelength, may be beyond a cut-off wavelength for a propagating mode inthe waveguide. According to some embodiments, the sample well 27-210 maycomprise a SCN for the excitation energy, such that the greatestintensity of excitation energy is localized to an excitation region27-215 of the sample well at an entrance to the sample well 27-210(e.g., localized near the interface between layer 27-235 and layer27-230 as depicted in the drawing). Such localization of the excitationenergy can improve localization of emission energy from the sample, andlimit the observed emission that emitted from a single sample (e.g., asingle molecule).

According to some embodiments, a pixel 27-100 may include additionalstructures. For example, a pixel 27-100 may include one or moreexcitation-coupling structure 27-220 that affects coupling of excitationenergy to a sample within the sample well. A pixel may also include anemission-directing structure 27-250 that affects directing emissionenergy from a sample within the sample well to sensor 27-260.

To improve the intensity of excitation energy that is localized at thesample well, other sample well structures were developed and studied bythe inventors. FIG. 27C depicts an embodiment of a sample well thatincludes a cavity or divot 27-216 at an excitation end of the samplewell. Adding a divot 27-216 to the sample well allows a sample to moveinto a region of higher excitation intensity, according to someembodiments. In some implementations, the shape and structure of thedivot alters the local excitation field (e.g., because of a differencein refractive index between the layer 27-235 and fluid in the samplewell), and can further increase the intensity of the excitation energyin the divot. Divot 27-216 may be formed within layer 27-235 such that aportion of the sample volume that occupies sample well 27-214 and divot27-216 is surrounded by the material that forms layer 27-216.

The divot may have any suitable shape. The divot may have a transverseshape that is substantially equivalent to a transverse shape of thesample well, e.g., round, elliptical, square, rectangular, polygonal,etc. In some embodiments, the sidewalls of the divot may besubstantially straight and vertical, like the walls of the sample well.In some implementations, the sidewalls of the divot may be sloped and/orcurved, as depicted in the drawing. The transverse dimension of thedivot may be approximately the same size as the transverse dimension ofthe sample well in some embodiments, may be smaller than the transversedimension of the sample well in some embodiments, or may be larger thanthe transverse dimension of the sample well in some embodiments. Thedivot 27-216 may extend between approximately 10 nm and approximately200 nm beyond sample well layer 27-230. In some implementations, thedivot may extend between approximately 50 nm and approximately 150 nmbeyond sample well layer 27-230. In some embodiments, the divot mayextend between approximately 150 nm and approximately 250 nm beyondsample well layer 27-230. By forming the divot, the excitation region27-215 may extend outside the sample well, as depicted in FIG. 27C.

Some embodiments relate to an integrated device having a sample wellwith a divot positioned proximate to a waveguide. FIG. 28 shows anintegrated device having sample well 28-632 formed in layer 28-630 andlayer 28-636. Layer 28-630 may be a metal layer and include one or moremetals (e.g., Al). Layer 28-636 may act as a dielectric layer andinclude one or more dielectric materials (e.g., silicon dioxide). Samplewell 28-632 may have a variable dimension in a direction parallel tolayer 28-630 and/or layer 28-636. Sample well 28-632 may have adimension D2 along the z-direction at least within layer 28-630 of theintegrated device, and in some embodiments, dimension D2 may beconsidered a diameter of sample well 28-632. Dimension D2 of sample well28-632 may be approximately 700 nm, approximately 800 nm, approximately900 nm, approximately 1 micron, or approximately 1.1 microns. Samplewell 28-632 may have a dimension D1 along the z-direction within layer28-636 of the integrated device and in some embodiments, may be considera diameter at a surface of sample well 28-632. Dimension D1 may beapproximately 100 nm, approximately 150 nm, approximately 200 nm, orapproximately 250 nm. The surface of sample well 28-632 having dimensionD1 is positioned a dimension dl along the x-direction from waveguide28-634. Positioning sample well 28-632 proximate to waveguide 28-634 bydistance dl may allow for improved coupling of excitation energy fromwaveguide 28-634 to sample well 28-632. Dimension dl may beapproximately 50 nm, approximately 100 nm, approximately 150 nm,approximately 200 nm, or approximately 250 nm.

D. Coupling Excitation Energy to Sample Well

Coupling of excitation energy to one or more sample wells of theintegrated device may occur through one or more techniques. Aspreviously discussed, in some embodiments, a waveguide is positioned tocouple with an excitation source to one or more sample wells. Asexcitation energy propagates along the waveguide, a portion of theexcitation energy may be couple to one or more sample wells through avariety of light coupling techniques. For example, the waveguide mayguide excitation energy substantially in one direction, and anevanescent wave or tail may form perpendicular to this one directionand, in some instances, be located outside the waveguide structure. Suchan evanescent tail may direct a portion of excitation energy towards oneor more sample wells. In some embodiments, the sample well layer may bedesigned and configured to direct excitation energy to a localizedregion within the sample well. The sample well may be configured toretain a sample within the localized region of the sample well such thatexcitation energy is directed towards the sample.

Additionally, one or more components may be formed in an integrateddevice to improve or enhance coupling of excitation energy into a samplewell. These additional components may be formed in a pixel and providecoupling of excitation energy from a waveguide into the pixel andtowards the sample well. One or more components located in a pixel mayact to tap a portion of the excitation energy from a waveguide into thepixel. Such components may include optical structures such as, gratingstructures, scattering structures, microcavities and/or nano-antennas.Features or configurations of one or more of these components may beselected for coupling a certain amount of excitation energy to eachsample well within a row or column of sample wells. A waveguideconfigured to provide excitation energy to a row of pixels may couple toa component in each pixel region to provide a portion of the excitationenergy to each pixel in the row of pixels. When a waveguide isconfigured to direct excitation energy from an excitation source towardsone or more pixels, the waveguide may be referred to as a bus waveguide.

Components positioned adjacent to a sample well may improve coupling ofexcitation energy from waveguide to sample well. Such components may becalled taps and/or microcavities. A microcavity may deflect a portion ofthe excitation energy from the waveguide such that excitation energyreaches the sample well. One or more microcavities may be used to coupleexcitation energy to a sample well. The one or more microcavities mayreduce loss of excitation energy from the waveguide, including metallicloss. One or more microcavities may act as a lens to focus excitationenergy to the sample well. In some embodiments, one or moremicrocavities may improve directing luminescence from a marker in thesample well towards the sensor. The microcavities may have acylindrical, convex, concave, rectangular, spherical, or ellipticalconfiguration or any other suitable shape. A microcavity may be formedfrom any suitable material. In some embodiments, a microcavity mayinclude silicon nitride.

One or more microcavities may overlap with at least a portion of awaveguide to direct excitation energy towards the sample well whenviewed from the top of the integrated device where the sample wells arepresent. The thickness of a waveguide may be configured to reduce lossof excitation energy and improve coupling of excitation energy to one ormore microcavities. In some embodiments, microcavities along a row ofsample wells may vary in strength of coupling between the waveguide toeach sample well. The microcavities may increase coupling along thepropagation direction of the excitation energy in order to accommodate areduced power in the waveguide as excitation energy is directed out ofthe waveguide to each sample well. In some embodiments, one or moremicrocavities are adjacent to the sample well. There may be an offsetdistance between the location of the center of a sample well and thecenter of a microcavity. In other embodiments, one microcavity islocated below a sample well such that at least a portion of the samplewell and a portion of the microcavity overlap when viewed from the topof the integrated device where the sample wells are present.

One or more dimensions of a waveguide of an integrated device may varyalong the length of the waveguide in the direction of light propagationthrough the waveguide. Varying one or more dimensions along thewaveguide may improve coupling efficiency and substantial uniformity inthe amount of excitation energy provided by the waveguide to a pluralityof sample wells. In some embodiments, the cross-sectional width of awaveguide may vary along a row or column of pixels. The waveguide mayinclude a taper such that the cross-sectional width of the waveguidedecreases along the direction of propagation of excitation energythrough the waveguide. In some embodiments, a tapered waveguide may beconfigured for a similar coupling efficiency for a row of pixels wherepixels in the row include a microcavity and a sample well. A combinationof varying one or more dimensions of the tapered waveguide and/or themicrocavity may accommodate reduction in power of excitation energyalong the length of a waveguide as excitation energy is coupled to eachsample well in the row.

In some embodiments, a waveguide may couple to a sample well by anevanescent field. In some embodiments, a bullseye grating structurehaving concentric grating rings positioned proximate to a sample wellmay improve coupling of excitation energy from the waveguide to thesample well. In some embodiments, the waveguide may include a regionhaving a reduced cross-sectional width in the vicinity of a sample wellsuch that an evanescent field from the excitation energy propagating inthe waveguide couples to the sample well. For a row of pixels, awaveguide may include multiple regions having a reduced cross-sectionalwidth along the length of the waveguide to improve coupling uniformityand efficiency among sample wells in the row. In this manner, thewaveguide may be considered to have pinched sections at certainlocations along the waveguide.

The layer of an integrated device having one or more sample wells mayinterfere with propagation of light through a waveguide. In someembodiments, the sample well layer is formed of a metal (e.g.,aluminum). It may be desirable to position the sample well layer at acertain distance from the waveguide to reduce loss and improveperformance of the device. These techniques may allow for a desiredperformance achieved by positioning the sample well layer at a certaindistance from the waveguide while allowing a sample well in the layer toreceive a sufficient amount of excitation energy.

E. Directing Emission Energy to Sensor

An integrated device may include one or more components positionedbetween a sample well and a sensor of a pixel to improve collection ofluminescence by the sensor from a sample in the sample well. Suchcomponents may improve the signal-to-noise ratio of the luminescencesignal to a background signal to provide improved detection of aluminescent marker. Some components may direct luminescence from asample well to a corresponding sensor in a pixel. In some embodiments, acomponent may provide both suitable coupling of excitation energy to asample well and coupling of luminescence out of the sample well. Othercomponents (e.g., filters) may reduce excitation energy and other lightnot associated with the sample and/or marker from contributing to asignal acquired by the sensor.

A bullseye grating may be formed from concentric grating rings around asample well. A bullseye grating may couple with a sample well to improvepropagation of luminescence out of the sample well. The bullseye gratingstructure may direct luminescence towards a sensor in a pixel having thesample well. In some embodiments, an effective diameter of theluminescence directed by a bullseye grating is approximately 5 microns.

In some embodiments, one or more microcavities provided for coupling ofa waveguide and a sample well to allow propagation of excitation energyto the sample well may also direct luminescence from the sample well toa sensor.

A baffle having an opening centered on a sensor may be formed between asample well and the sensor. As shown in FIG. 26, baffle layer 26-226 ispositioned between sample well 26-22 and sensor layer 26-230. A bafflein a pixel may be designed to suppress collection of energy besidesluminescence for the pixel. A baffle associated with a sample well and asensor may allow for luminescence from the sample well to reach thesensor while reducing luminescence from neighboring pixels, excitationenergy, and other energy not associated with the luminescence from thesample well associated with the sensor. A dimension of the opening ofthe baffle may be configured to allow luminescence directed by abullseye on the same pixel. As shown in FIG. 26, baffle 26-226 hasopenings along the z-direction to allow luminescence to pass through tosensors positioned in layer 26-230. The material of a baffle may beselected for certain optical properties, such as reducing transmissionof certain light wavelengths or energies at certain incident angles. Insome embodiments, a baffle may be formed by multiple layers of materialshaving different refractive indices. A baffle may include one or morelayers of silicon, silicon nitride (Si3N4), silicon, titanium nitride(TiN), and aluminum (Al). A layer configured to form a baffle may have across-sectional height of approximately 20 nm, approximately 20 nm,approximately 50 nm, approximately 60 nm, approximately 70 nm,approximately 80 nm, or approximately 90 nm.

Filtering components may be formed between a waveguide and a sensor forreducing collection of excitation energy by the sensor. Any suitablemanner for filtering excitation energy may be provided. Techniques forfiltering the excitation energy may include filtering based on one ormore characteristics of light. Such characteristics may includewavelength and/or polarization. Filtering components may selectivelysuppress scattered excitation energy while allowing luminescence to passthrough to the sensor. Layer 26-228 shown in FIG. 26 may include one ormore filtering components described herein.

An integrated device may include a wavelength filter configured toreflect light of one or more characteristic wavelengths and allowtransmission of light having a different characteristic wavelength. Insome embodiments, light reflected by a wavelength filter may have ashorter characteristic wavelength than the light transmitted by thewavelength filter. Such a wavelength filter may reflect excitationenergy and allow transmission of luminesce since excitation energy usedto excite a marker typically has a shorter characteristic wavelengththan luminesce emitted by the marker in response reaching an excitationstate by the excitation energy.

F. Sensor

Any suitable sensor capable of acquiring time bin information may beused for measurements to detect lifetimes of luminescent markers. Forexample, U.S. patent application Ser. No. 14/821,656 entitled“INTEGRATED DEVICE FOR TEMPORAL BINNING OF RECEIVED PHOTONS,” describesa sensor capable of determining an arrival time of a photon, and isincorporated by reference in its entirety. The sensors are aligned suchthat each sample well has at least one sensor region to detectluminescence from the sample well. In some embodiments, the integrateddevice may include Geiger mode avalanche photodiode arrays and/or singlephoton avalanche diode arrays (SPADs).

Described herein is an integrated photodetector that can accuratelymeasure, or “time-bin,” the timing of arrival of incident photons, andwhich may be used in a variety of applications, such as sequencing ofnucleic acids (e.g., DNA sequencing), for example. In some embodiments,the integrated photodetector can measure the arrival of photons withnanosecond or picosecond resolution, which can facilitate time-domainanalysis of the arrival of incident photons.

Some embodiments relate to an integrated circuit having a photodetectorthat produces charge carriers in response to incident photons and whichis capable of discriminating the timing at which the charge carriers aregenerated by the arrival of incident photons with respect to a referencetime (e.g., a trigger event). In some embodiments, a charge carriersegregation structure segregates charge carriers generated at differenttimes and directs the charge carriers into one or more charge carrierstorage regions (termed “bins”) that aggregate charge carriers producedwithin different time periods. Each bin stores charge carriers producedwithin a selected time interval. Reading out the charge stored in eachbin can provide information about the number of photons that arrivedwithin each time interval. Such an integrated circuit can be used in anyof a variety of applications, such as those described herein.

An example of an integrated circuit having a photodetection region and acharge carrier segregation structure will be described. In someembodiments, the integrated circuit may include an array of pixels, andeach pixel may include one or more photodetection regions and one ormore charge carrier segregation structures, as discussed below.

FIG. 29A shows a diagram of a pixel 29-900, according to someembodiments. Pixel 29-900 includes a photon absorption/carriergeneration region 29-902 (also referred to as a photodetection region),a carrier travel/capture region 29-906, a carrier storage region 29-908having one or more charge carrier storage regions, also referred toherein as “charge carrier storage bins” or simply “bins,” and readoutcircuitry 29-910 for reading out signals from the charge carrier storagebins.

The photon absorption/carrier generation region 29-902 may be a regionof semiconductor material (e.g., silicon) that can convert incidentphotons into photogenerated charge carriers. The photonabsorption/carrier generation region 29-902 may be exposed to light, andmay receive incident photons. When a photon is absorbed by the photonabsorption/carrier generation region 29-902 it may generatephotogenerated charge carriers, such as an electron/hole pair.Photogenerated charge carriers are also referred to herein simply as“charge carriers.”

An electric field may be established in the photon absorption/carriergeneration region 29-902. In some embodiments, the electric field may be“static,” as distinguished from the changing electric field in thecarrier travel/capture region 29-906. The electric field in the photonabsorption/carrier generation region 29-902 may include a lateralcomponent, a vertical component, or both a lateral and a verticalcomponent. The lateral component of the electric field may be in thedownward direction of FIG. 29A, as indicated by the arrows, whichinduces a force on photogenerated charge carriers that drives themtoward the carrier travel/capture region 106. The electric field may beformed in a variety of ways.

In some embodiments, one or more electrodes may be formed over thephoton absorption/carrier generation region 29-902. The electrodes(s)may have voltages applied thereto to establish an electric field in thephoton absorption/carrier generation region 29-902. Such electrode(s)may be termed “photogate(s).” In some embodiments, photonabsorption/carrier generation region 29-902 may be a region of siliconthat is fully depleted of charge carriers.

In some embodiments, the electric field in the photon absorption/carriergeneration region 29-902 may be established by a junction, such as a PNjunction. The semiconductor material of the photon absorption/carriergeneration region 29-902 may be doped to form the PN junction with anorientation and/or shape that produces an electric field that induces aforce on photogenerated charge carriers that drives them toward thecarrier travel/capture region 29-906. In some embodiments, the Pterminal of the PN junction diode may connected to a terminal that setsits voltage. Such a diode may be referred to as a “pinned” photodiode. Apinned photodiode may promote carrier recombination at the surface, dueto the terminal that sets its voltage and attracts carriers, which canreduce dark current. Photogenerated charge carriers that are desired tobe captured may pass underneath the recombination area at the surface.In some embodiments, the lateral electric field may be established usinga graded doping concentration in the semiconductor material.

As illustrated in FIG. 29A, a photon may be captured and a chargecarrier 29-901A (e.g., an electron) may be produced at time t1. In someembodiments, an electrical potential gradient may be established alongthe photon absorption/carrier generation region 29-902 and the carriertravel/capture region 29-906 that causes the charge carrier 29-901A totravel in the downward direction of FIG. 29A (as illustrated by thearrows shown in FIG. 29A). In response to the potential gradient, thecharge carrier 29-901A may move from its position at time t1 to a secondposition at time t2, a third position at time t3, a fourth position attime t4, and a fifth position at time t5. The charge carrier 29-901Athus moves into the carrier travel/capture region 29-906 in response tothe potential gradient.

The carrier travel/capture region 29-906 may be a semiconductor region.In some embodiments, the carrier travel/capture region 29-906 may be asemiconductor region of the same material as photon absorption/carriergeneration region 29-902 (e.g., silicon) with the exception that carriertravel/capture region 29-906 may be shielded from incident light (e.g.,by an overlying opaque material, such as a metal layer).

In some embodiments, and as discussed further below, a potentialgradient may be established in the photon absorption/carrier generationregion 29-902 and the carrier travel/capture region 29-906 by electrodespositioned above these regions. However, the techniques described hereinare not limited as to particular positions of electrodes used forproducing an electric potential gradient. Nor are the techniquesdescribed herein limited to establishing an electric potential gradientusing electrodes. In some embodiments, an electric potential gradientmay be established using a spatially graded doping profile. Any suitabletechnique may be used for establishing an electric potential gradientthat causes charge carriers to travel along the photonabsorption/carrier generation region 29-902 and carrier travel/captureregion 29-906.

A charge carrier segregation structure may be formed in the pixel toenable segregating charge carriers produced at different times. In someembodiments, at least a portion of the charge carrier segregationstructure may be formed over the carrier travel/capture region 29-906.As will be described below, the charge carrier segregation structure mayinclude one or more electrodes formed over the carrier travel/captureregion 29-906, the voltage of which may be controlled by controlcircuitry to change the electric potential in the carrier travel/captureregion 29-906.

The electric potential in the carrier travel/capture region 29-906 maybe changed to enable capturing a charge carrier. The potential gradientmay be changed by changing the voltage on one or more electrodesoverlying the carrier travel/capture region 29-906 to produce apotential barrier that can confine a carrier within a predeterminedspatial region. For example, the voltage on an electrode overlying thedashed line in the carrier travel/capture region 29-906 of FIG. 29A maybe changed at time t5 to raise a potential barrier along the dashed linein the carrier travel/capture region 29-906 of FIG. 29A, therebycapturing charge carrier 29-901A. As shown in FIG. 29A, the carriercaptured at time t5 may be transferred to a bin “bin0” of carrierstorage region 29-908. The transfer of the carrier to the charge carrierstorage bin may be performed by changing the potential in the carriertravel/capture region 29-906 and/or carrier storage region 29-908 (e.g.,by changing the voltage of electrode(s) overlying these regions) tocause the carrier to travel into the charge carrier storage bin.

Changing the potential at a certain point in time within a predeterminedspatial region of the carrier travel/capture region 29-906 may enabletrapping a carrier that was generated by photon absorption that occurredwithin a specific time interval. By trapping photogenerated chargecarriers at different times and/or locations, the times at which thecharge carriers were generated by photon absorption may bediscriminated. In this sense, a charge carrier may be “time binned” bytrapping the charge carrier at a certain point in time and/or spaceafter the occurrence of a trigger event. The time binning of a chargecarrier within a particular bin provides information about the time atwhich the photogenerated charge carrier was generated by absorption ofan incident photon, and thus likewise “time bins,” with respect to thetrigger event, the arrival of the incident photon that produced thephotogenerated charge carrier.

FIG. 29B illustrates capturing a charge carrier at a different point intime and space. As shown in FIG. 29B, the voltage on an electrodeoverlying the dashed line in the carrier travel/capture region 29-906may be changed at time t9 to raise a potential barrier along the dashedline in the carrier travel/capture region 106 of FIG. 29B, therebycapturing carrier 29-901B. As shown in FIG. 29B, the carrier captured attime t9 may be transferred to a bin “bin1” of carrier storage region29-908. Since charge carrier 29-901B is trapped at time t9, itrepresents a photon absorption event that occurred at a different time(i.e., time t6) than the photon absorption event (i.e., at t1) forcarrier 29-901A, which is captured at time t5.

Performing multiple measurements and aggregating charge carriers in thecharge carrier storage bins of carrier storage region 29-908 based onthe times at which the charge carriers are captured can provideinformation about the times at which photons are captured in the photonabsorption/carrier generation area 29-902. Such information can beuseful in a variety of applications, as discussed above.

In some embodiments, the duration of time each time bin captures afteran excitation pulse may vary. For example, shorter time bins may be usedto detect luminescence shortly after the excitation pulse, while longertime bins may be used at times further from an excitation pulse. Byvarying the time bin intervals, the signal to noise ratio formeasurements of the electrical signal associated with each time bin maybe improved for a given sensor. Since the probability of a photonemission event is higher shortly after an excitation pulse, a time binwithin this time may have a shorter time interval to account for thepotential of more photons to detect. While at longer times, theprobability of photon emission may be less and a time bin detectingwithin this time may be longer to account for a potential fewer numberof photons. In some embodiments, a time bin with a significantly longertime duration may be used to distinguish among multiple lifetimes. Forexample, the majority of time bins may capture a time interval in therange of approximately 0.1-0.5 ns, while a time bin may capture a timeinterval in the range of approximately 2-5 ns. The number of time binsand/or the time interval of each bin may depend on the sensor used todetect the photons emitted from the sample object.

Determining the time interval for each bin may include identifying thetime intervals needed for the number of time bins provided by the sensorto distinguish among luminescent markers used for analysis of a sample.The distribution of the recorded histogram may be compared to knownhistograms of markers under similar conditions and time bins to identifythe type of marker in the sample well. Different embodiments of thepresent application may measure lifetimes of markers but vary in theexcitation energies used to excite a marker, the number of sensorregions in each pixel, and/or the wavelength detected by the sensors.

III. Excitation Source

According to some embodiments, one or more excitation sources may belocated external to the integrated device, and may be arranged todeliver pulses of light to an integrated device having sample wells. Forexample, U.S. Provisional Patent Application 62/164,485, entitled“PULSED LASER,” filed May 20, 2015 and U.S. Provisional PatentApplication 62/310,398, entitled “PULSED LASER,” filed Mar. 18, 2016describe examples of pulsed laser sources that may be used as anexcitation source, each of which is incorporated by reference in itsentirety. The pulses of light may be coupled to a plurality of samplewells and used to excite one or more markers within the wells, forexample. The one or more excitation sources may deliver pulses of lightat one or more characteristic wavelengths, according to someimplementations. In some cases, an excitation source may be packaged asan exchangeable module that mounts in or couples to a base instrument,into which the integrated device may be loaded. Energy from anexcitation source may be delivered radiatively or non-radiatively to atleast one sample well or to at least one sample in at least one samplewell. In some implementations, an excitation source having acontrollable intensity may be arranged to deliver excitation energy to aplurality of pixels of an integrated device. The pixels may be arrangedin a linear array (e.g., row or column), or in a 2D array (e.g., asub-area of the array of pixels or the full array of pixels).

Any suitable light source may be used for an excitation source. Someembodiments may use incoherent light sources and other embodiments mayuse coherent light sources. By way of non-limiting examples, incoherentlight sources according to some embodiments may include different typesof light emitting diodes (LEDs) such as organic LEDs (OLEDs), quantumdots (QLEDs), nanowire LEDs, and (in)organic semiconductor LEDs. By wayof non-limiting examples, coherent light sources according to someembodiments may include different types of lasers such as semiconductorlasers (e.g., vertical cavity surface emitting lasers (VCSELs), edgeemitting lasers, and distributed-feedback (DFB) laser diodes).Additionally or alternatively, slab-coupled optical waveguide laser(SCOWLs) or other asymmetric single-mode waveguide structures may beused. In some implementations, coherent light sources may compriseorganic lasers, quantum dot lasers, and solid state lasers (e.g., aNd:YAG or ND:Glass laser, pumped by laser diodes or flashlamps). In someembodiments, a laser-diode-pumped fiber laser may be used. A coherentlight source may be passively mode locked to produce ultrashort pulses.There may be more than one type of excitation source for an array ofpixels on an integrated device. In some embodiments, different types ofexcitation sources may be combined. An excitation source may befabricated according to conventional technologies that are used tofabricate a selected type of excitation source.

By way of introduction and without limiting the invention, an examplearrangement of a coherent light source is depicted in FIG. 30A. Thedrawing illustrates an analytical instrument 30-100 that may include anultrashort-pulsed laser excitation source 30-110 as the excitationsource. The ultrashort pulsed laser 30-110 may comprise a gain medium30-105 (which may be a solid-state material is some embodiments), a pumpsource for exciting the gain medium (not shown), and at least two cavitymirrors 30-102, 30-104 that define ends of an optical laser cavity. Insome embodiments, there may be one or more additional optical elementsin the laser cavity for purposes of beam shaping, wavelength selection,and/or pulse forming. When operating, the pulsed-laser excitation source30-110 may produce an ultrashort optical pulse 30-120 that circulatesback-and-forth in the laser cavity between the cavity's end mirrors30-102, 30-104 and through the gain medium 30-105. One of the cavitymirrors 30-104 may partially transmit a portion of the circulatingpulse, so that a train of optical pulses 30-122 is emitted from thepulsed laser 30-110 to subsequent component 30-160, such as an opticalcomponent and integrated device. The emitted pulses may sweep out a beam(indicated by the dashed lines) that is characterized by a beam waist w.

Measured temporal intensity profiles of the emitted pulses 30-122 mayappear as depicted in FIG. 30B. In some embodiments, the peak intensityvalues of the emitted pulses may be approximately equal, and theprofiles may have a Gaussian temporal profile, though other profilessuch as a sech profile may be possible. In some cases, the pulses maynot have symmetric temporal profiles and may have other temporal shapes.In some embodiments, gain and/or loss dynamics may yield pulses havingasymmetric profiles. The duration of each pulse may be characterized bya full-width-half-maximum (FWHM) value, as indicated in FIG. 30B.Ultrashort optical pulses may have FWHM values less than 100picoseconds.

The pulses emitting from a laser excitation source may be separated byregular intervals T. In some embodiments, T may be determined by activegain and/or loss modulation rates in the laser. For mode-locked lasers,T may be determined by a round-trip travel time between the cavity endmirrors 30-102, 30-104. According to some embodiments, the pulseseparation time T may be between about 1 ns and about 100 ns. In somecases, the pulse separation time T may be between about 0.1 ns and about1 ns. In some implementations, the pulse separation time T may bebetween about 100 ns and about 2 μs.

In some embodiments, an optical system 30-140 may operate on a beam ofpulses 30-122 from a laser excitation source 30-110. For example, theoptical system may include one or more lenses to reshape the beam and/orchange the divergence of the beam. Reshaping of the beam may includeincreasing or decreasing the value of the beam waist and/or changing across-sectional shape of the beam (e.g., elliptical to circular,circular to elliptical, etc.). Changing the divergence of the beam maycomprise converging or diverging the beam flux. In some implementations,the optical system 30-140 may include an attenuator or amplifier tochange the amount of beam energy. In some cases, the optical system mayinclude wavelength filtering elements. In some implementations, theoptical system may include pulse shaping elements, e.g., a pulsestretcher and/or pulse compressor. In some embodiments, the opticalsystem may include one or more nonlinear optical elements, such as asaturable absorber for reducing a pulse length. According to someembodiments, the optical system 30-140 may include one or more elementsthat alter the polarization of pulses from a laser excitation source30-110. In some implementations, an optical system 30-140 may include anonlinear crystal for converting the output wavelength from anexcitation source 30-110 to a shorter wavelength via frequency doublingor to a longer wavelength via parametric amplification. For example, anoutput of the laser may be frequency-doubled in a nonlinear crystal(e.g., in periodically-poled lithium niobate (PPLN)) or other non-polednonlinear crystal. Such a frequency-doubling process may allow moreefficient lasers to generate wavelengths more suitable for excitation ofselected fluorophores.

The phrase “characteristic wavelength” or “wavelength” may refer to acentral or predominant wavelength within a limited bandwidth ofradiation produced by an excitation source. In some cases, it may referto a peak wavelength within a bandwidth of radiation produced by anexcitation source. A characteristic wavelength of an excitation sourcemay be selected based upon a choice of luminescent markers or probesthat are used in a bioanalysis device, for example. In someimplementations, the characteristic wavelength of a source of excitationenergy is selected for direct excitation (e.g., single photonexcitation) of a chosen fluorophore. In some implementations, thecharacteristic wavelength of an excitation source is selected forindirect excitation (e.g., multi-photon excitation or harmonicconversion to a wavelength that will provide direct excitation). In someembodiments, excitation radiation may be generated by a light sourcethat is configured to generate excitation energy at a particularwavelength for application to a sample well. In some embodiments, acharacteristic wavelength of the excitation source may be less than acharacteristic wavelength of corresponding emission from the sample. Forexample, an excitation source may emit radiation having a characteristicwavelength between 500 nm and 700 nm (e.g., 515 nm, 532 nm, 563 nm, 594nm, 612 nm, 632 nm, 647 nm). In some embodiments, an excitation sourcemay provide excitation energy centered at two different wavelengths,such as 532 nm and 593 nm for example.

In some embodiments, a pulsed excitation source may be used to excite aluminescent marker in order to measure an emission lifetime of theluminescent marker. This can be useful for distinguishing luminescentmarkers based on emission lifetime rather than emission color orwavelength. As an example, a pulsed excitation source may periodicallyexcite a luminescent marker in order to generate and detect subsequentphoton emission events that are used to determine a lifetime for themarker. Lifetime measurements of luminescent markers may be possiblewhen the excitation pulse from an excitation source transitions from apeak pulse power or intensity to a lower (e.g., nearly extinguished)power or intensity over a duration of time that is less than thelifetime of the luminescent marker. It may be beneficial if theexcitation pulse terminates quickly, so that it does not re-excite theluminescent marker during a post-excitation phase when a lifetime of theluminescent marker is being evaluated. By way of example and notlimitation, the pulse power may drop to approximately 20 dB,approximately 40 dB, approximately 80 dB, or approximately 120 dB lessthan the peak power after 250 picoseconds. In some implementations, thepulse power may drop to approximately 20 dB, approximately 40 dB,approximately 80 dB, or approximately 120 dB less than the peak powerafter 100 picoseconds.

An additional advantage of using ultrashort excitation pulses to exciteluminescent markers is to reduce photo-bleaching of the markers.Applying continuous excitation energy to a marker may bleach and/ordamage a luminescent marker over time. Even though a peak pulse power ofthe excitation source may be considerably higher than a level that wouldrapidly damage a marker at continuous exposure, the use of ultrashortpulses may increase the amount of time and number of useful measurementsbefore the marker becomes damaged by the excitation energy.

When using a pulsed excitation source to discern lifetimes ofluminescent markers, the time between pulses of excitation energy may beas long as or longer than a longest lifetime of the markers in order toobserve and evaluate emission events after each excitation pulse. Forexample, the time interval T (see FIG. 30B) between excitation pulsesmay be longer than any emission lifetime of the examined fluorophores.In this case, a subsequent pulse may not arrive before an excitedfluorophore from a previous pulse has had a reasonable amount of time tofluoresce. In some embodiments, the interval T needs to be long enoughto determine a time between an excitation pulse that excites afluorophore and a subsequent photon emitted by the fluorophore aftertermination of excitation pulse and before the next excitation pulse.

Although the interval between excitation pulses T should be long enoughto observe decay properties of the fluorophores, it is also desirablethat T is short enough to allow many measurements to be made in a shortperiod of time. By way of example and not limitation, emission lifetimesof fluorophores used in some applications may be in the range of about100 picoseconds to about 10 nanoseconds. Accordingly, excitation pulsesused to detect and/or discern such lifetimes may have durations (FWHM)ranging from about 25 picoseconds to about 2 nanoseconds, and may beprovided at pulse repetition rates ranging from about 20 MHz to about 1GHz.

In further detail, any suitable techniques for modulating the excitationenergy to create a pulsed excitation source for lifetime measurementsmay be used. Direct modulation of an excitation source, such as a laser,may involve modulating the electrical drive signal of the excitationsource so the emitted power is in the form of pulses. The input powerfor a light source, including the optical pumping power, andexcited-state carrier injection and/or carrier removal from a portion ofthe gain region, may be modulated to affect the gain of the gain medium,allowing the formation of pulses of excitation energy through dynamicgain shaping. Additionally, the quality (Q) factor of the opticalresonator may be modulated by various means to form pulses usingQ-switching techniques. Such Q-switching techniques may be active and/orpassive. The longitudinal modes of a resonant cavity of a laser may bephase-locked to produce a series of pulses of emitted light throughmode-locking. Such mode-locking techniques may be active and/or passive.A laser cavity may include a separate absorbing section to allow formodulation of the carrier density and control of the absorption loss ofthat section, thus providing additional mechanisms for shaping theexcitation pulse. In some embodiments, an optical modulator may be usedto modulate a beam of continuous wave (CW) light to be in the form of apulse of excitation energy. In other embodiments, a signal sent to anacoustic optic modulator (AOM) coupled to an excitation source may beused to change the deflection, intensity, frequency, phase, and/orpolarization of the outputted light to produce a pulsed excitationenergy. AOMs may also be used for continuous wave beam scanning,Q-switching, and/or mode-locking. Although the above techniques aredescribed for creating a pulsed excitation source, any suitable way toproduce a pulsed excitation source may be used for measuring lifetimesof luminescent markers.

In some embodiments, techniques for forming a pulsed excitation sourcesuitable for lifetime measurements may include modulation of an inputelectrical signal that drives photon emission. Some excitation sources(e.g., diode lasers and LEDs) convert an electrical signal, such as aninput current, into a light signal. The characteristics of the lightsignal may depend on characteristics of the electrical signal. Inproducing a pulsed light signal, the electrical signal may vary overtime in order to produce a variable light signal. Modulating theelectrical signal to have a specific waveform may produce an opticalsignal with a specific waveform. The electrical signal may have asinusoidal waveform with a certain frequency and the resulting pulses oflight may occur within time intervals related to the frequency. Forexample, an electrical signal with a 500 MHz frequency may produce alight signal with pulses every 2 nanoseconds. Combined beams produced bydistinct pulsed excitation sources, whether similar or different fromeach other, may have a relative path difference below 1 mm.

In some excitation sources, such as laser diodes, the electrical signalchanges the carrier density and photons are produced through therecombination of electron and hole pairs. The carrier density is relatedto the light signal such that when the carrier density is above athreshold, a substantial number of coherent photons are generated viastimulated emission. Current supplied to a laser diode may injectelectrons or carriers into the device, and thereby increase the carrierdensity. When the carrier density is above a threshold, photons may begenerated at a faster rate than the current supplying the carriers, andthus the carrier density may decrease below the threshold and photongeneration reduces. With the photon generation reduced, the carrierdensity begins to increase again, due to continued current injection andthe absorption of photons, and eventually increases above the thresholdagain. This cycle leads to oscillations of the carrier density aroundthe threshold value for photon generation, resulting in an oscillatinglight signal. These dynamics, known as relaxation oscillations, can leadto artifacts in the light signal due to oscillations of the carrierdensity. When current is initially supplied to a laser, there may beoscillations before the light signal reaches a stable power due tooscillations in the carrier density. When forming a pulsed excitationsource, oscillations of the carrier density may introduce artifacts fora pulsed light signal. Artifacts from such relaxation oscillations maybroaden a pulsed light signal and/or produce a tail in the light signal,limiting the lifetimes that can be detected by such a pulsed lightsource since the excitation signal may overlap with emitted photons by aluminescent marker.

In some embodiments, techniques for shortening the time duration of anexcitation pulse may be used to reduce the excitation energy required todetect luminescent markers and thereby reduce or delay bleaching andother damage to the luminescent markers. Techniques for shortening thetime duration of the excitation pulse may be used to reduce the powerand/or intensity of the excitation energy after a maximum value or peakof the excitation pulse, allowing the detection of shorter lifetimes.Such techniques may electrically drive the excitation source in order toreduce the excitation power after the peak power. An electrical drivingsignal may be tailored to drive the intensity of the pulse of excitationenergy to zero as quickly as possible after the peak pulse. Such atechnique may involve reversing the sign of an electrical driving signalafter the peak power is produced. The electrical signal may be tailoredto quickly reduce the carrier density after the first relaxationoscillation or first oscillation of the optical signal. By reducing thecarrier density after the first oscillation, a light pulse of just thefirst oscillation may be generated. The electrical signal may beconfigured to generate a short pulse that turns the light signal offquickly by reducing the number of photons emitted after a peak in thesignal. A picosecond laser diode system may be designed to emit lightpulses, according to some embodiments. In some embodiments, saturableabsorbers, including semiconductor saturable absorbers (SESAMs) may beused to suppress the optical tail. In such embodiments, using thesaturable absorbers may suppress the optical tail by 3-5 dB or, in someinstances, greater than 5 dB. Reducing the effects of a tail in theexcitation pulse may reduce and/or eliminate any requirements onadditional filtering of the excitation energy, increase the range oflifetimes that can be measured, and/or enable faster pulse rates.Increasing the excitation pulse rate may enable more experiments to beconducted in a given time, which may decrease the time needed to acquireenough statistics to identify a lifetime for a marker labeling a sampleobject.

Additionally, two or more of these techniques may be used together togenerate pulsed excitation energy. For example, pulsed excitation energyemitted from a directly modulated source may be further modified usingoptical modulation techniques. Techniques for modulating the excitationpulse and tailoring the electrical pulse driving signal may be combinedin any suitable way to optimize a pulsed excitation energy forperforming lifetime measurements. A tailored electrical drive signal maybe applied to a pulsed excitation energy from a directly modulatedsource.

In some embodiments, a laser diode having a certain number of wire bondsmay be used as a pulsed excitation source. Laser diodes with more wirebonds may reduce the inductance of the excitation source. Laser diodeshaving a lower inductance may enable the current into the laser tooperate at a higher frequency. Selecting a packaging method to minimizeinductance may improve the power supplied to the excitation source athigher frequencies, enabling shorter excitation pulses, fasterreductions of optical power after the peak, and/or increased pulserepetition rate for detecting luminescent markers.

In some embodiments, a transmission line in combination with anexcitation source may be used for generating light pulses. Thetransmission line may match the impedance of a laser diode in order toimprove performance and/or quality of light pulses. In some embodiments,the transmission line impedance may be 50 ohms. In some instances, theterminating resistance may be similar to the resistance of the line inorder to avoid reflections. Alternatively or additionally, theterminating impedance may be similar to the impedance of the line inorder to avoid reflections. The terminating impedance may less than theimpedance of the line in order to reflect a negative pulse. In otherembodiments, the terminating impedance may have a capacitive orinductive component in order to control the shape of the negativereflection pulse. In other embodiments, the transmission line may allowfor a higher frequency of pulses. Using a transmission line may produceelectrical pulses having a frequency within a range of 40 MHz to 500MHz. A transmission line may be used in combination with a tailoredelectrical signal described above in order to produce a pulsed lightsource with light pulses having a certain time duration and a specifictime interval.

Techniques for tailoring the electrical signal to improve the productionof light pulses may include connecting the excitation source to acircuit with a negative bias capability. In some embodiments, a negativebias may be provided on an excitation source after a light pulse emitsto reduce emission of a tail in the light pulse. An exemplary circuitmay include a current source, diode laser, resistor, capacitor, andswitch that may be implemented to reduce the presence of a tail in alight pulse. Such a circuit may create a constant current that bypassesthe diode laser when the switch is closed, or in a conducting state.When the switch is open, the switch may have a high resistance andcurrent may flow through the diode laser. Light pulses may be generatedby opening and closing the switch to provide intermittent current to thediode laser. In some instances, the resistor may be sufficiently highand the capacitor sufficiently small such that there is a voltage acrossthe capacitor when the switch is open and the diode laser emits light.When the switch is closed, the voltage across the capacitor will reversebias the diode laser. Such a reverse bias may reduce or eliminate thepresence of a tail in the light pulse. In such instances, the switch maybe configured to close after the peak of the light pulse in order toreduce the laser power shortly after the peak light pulse. The value ofthe resistor in the circuit may be selected such that the charge on thecapacitor will discharge before the switch is subsequently opened and/ora subsequent light pulse is generated by the laser diode.

Additional circuit components may be provided to tailor an electricalsignal of a laser diode in order to produce light pulses. In someembodiments, multiple capacitors, resistors, and voltages may beconnected as a network circuit to control the waveform of an electricalsignal supplied to a laser diode. A controlled waveform may be createdby switching a number of voltages, V1, V2, . . . , VN with correspondingsignals S1, S2, . . . , SN when there are N capacitor sub-circuits.

In some embodiments, an electrical signal for generating light pulsesmay use a circuit having discrete components, including radio frequency(RF) and/or microwave components. Discrete components that may beincluded in such a circuit are DC blocks, adaptors, logic gates,terminators, phase shifters, delays, attenuators, combiners, and/or RFamplifiers. Such components may be used to create a positive electricalsignal having a certain amplitude followed by a negative electricalsignal with another amplitude. There may be a delay between the positiveand negative electrical signals. In other embodiments, a circuit mayproduce multiple electrical signals combined to form an electrical pulsesignal configured to drive an excitation source. Such a circuit mayproduce a differential output which may be used to increase the power ofthe light pulse. By adjusting the discrete components of the circuit,the electrical output signal may be adjusted to produce a light pulsesuitable for lifetime measurements.

In some embodiments, excitation sources may be combined to generatelight pulses for lifetime measurements. Synchronized pulsed sources maybe coupled to a circuit or load over a certain distance. In someembodiments, excitation sources may be coupled in parallel to a circuit.The excitation sources may be from the same source or from multiplesources. In some embodiments with multiple sources, the multiple sourcesmay vary in type of excitation source. When combining sources, it may beimportant to consider the impedance of the circuit and the excitationsources in order to have sufficient power supplied to the excitationsources. Combination of sources may be achieved by using one or more ofthe above-described techniques for producing a pulsed excitation source.

An excitation source may include a battery or any other power supplyarranged to provide power to the excitation source. For example, anexcitation source may be located in a base instrument and its operatingpower may be received through an integrated bioanalysis device to whichit is coupled (e.g., via conducting power leads). An excitation sourcemay be controlled independently of or in collaboration with control ofan integrated bioanalysis device. As just one example, control signalsfor an excitation source may be provided to the excitation sourcewirelessly or via a wired interconnection (e.g., a USB interconnect)with a personal computer and/or the integrated bioanalysis device.

In some implementations, an excitation source may be operated in atime-gated and/or synchronized manner with one or more sensors of anintegrated device. For example, an excitation source may be turned on toexcite a luminescent marker, and then turned off. The sensor may beturned off while the excitation source is turned on, and then may beturned on for a sampling interval after the excitation source is turnedoff. In some embodiments, the sensor may be turned on for a samplinginterval while the excitation source is turned on.

IV. Example Measurements with the Integrated Device and ExcitationSource

Measurements for detecting, analyzing, and/or probing molecules in asample may be obtained using any combination of the integrated device orintegrated device and an excitation source described in the presentapplication. The excitation source may be a pulsed excitation source or,in some instances, a continuous wave source. A luminescent marker taggedto a specific sample may indicate the presence of the sample.Luminescent markers may be distinguished by an excitation energy,luminescence emission wavelength, and/or the lifetime of emission energyemitted by a marker. Markers with similar luminescence emissionwavelength may be identified by determining the lifetime for eachmarker. Additionally, markers with similar lifetimes may be identifiedby the luminescence emission wavelength for each marker. By usingmarkers, where markers are identified by a combination of the temporaland/or spectral properties of the emitted luminescence, a quantitativeanalysis and/or identification of markers and associated samples may beperformed.

Lifetime measurements may be used to determine that a marker is presentin a sample well. The lifetime of a luminescent marker may be identifiedby performing multiple experiments where the luminescent marker isexcited into an excited state and then the time when a photon emits ismeasured. The excitation source is pulsed to produce a pulse ofexcitation energy and directed at the marker. The time between theexcitation pulse and a subsequent photon emission event from aluminescent marker is measured. By repeating such experiments with aplurality of excitation pulses, the number of instances a photon emitswithin a particular time interval may be determined. Such results maypopulate a histogram representing the number of photon emission eventsthat occur within a series of discrete time intervals or time bins. Thenumber of time bins and/or the time interval of each bin may be adjustedto identify a particular set of lifetimes and/or markers.

What follows is a description of example measurements that may be madeto identify luminescent markers in some embodiments. Specifically,examples of distinguishing luminescent markers using only a luminescentlifetime measurement, a joint spectral and luminescent lifetimemeasurement, and only a luminescent lifetime measurement, but using twodifferent excitation energies are discussed. Embodiments are not limitedto the examples detailed below. For example, some embodiments mayidentify the luminescent markers using only spectral measurements.

Any suitable luminescent markers may be used. In some embodiments,commercially available fluorophores may be used. By way of example andnot limitation, the following fluorophores may be used: Atto Rho14(“ATRho14”), DyLight® 650 (“D650”), Seta™Tau 647 (“ST647”), CF™ 633(“C633”), CF™ 647 (“C647”), Alexa Fluor® 647 (“AF647”), BODIPY® 630/650(“B630”), CF™ 640R (“C640R”) and/or Atto 647N (“AT647N”).

Additionally and/or optionally, luminescent markers may be modified inany suitable way to increase the speed and accuracy of the sampleanalysis process. For example, a photostabilizer may be conjugated to aluminescent marker. Examples of photostabilizers include but are notlimited to oxygen scavengers or triplet-state quenchers. Conjugatingphotostabilizers to the luminescent marker may increase the rate ofphotons emitted and may also reduce a “blinking” effect where theluminescent marker does not emit photons. In some embodiments, when abiological event occurs on the millisecond scale, an increased rate ofphoton emission may increase the probability of detection of thebiological event. Increased rates of photon events may subsequentlyincrease the signal-to-noise ratio of luminescence signal and increasethe rate at which lifetime measurements are made, leading to a fasterand more accurate sample analysis.

Furthermore, the environment in a sample well of an integrated devicemay be tuned to engineer the lifetime of the markers as needed. This canbe achieved by recognizing that the lifetime of a marker is effected bythe density of state of the marker, which can be tuned using theenvironment. For example, the farther a marker is from the bottom metallayer of the sample well, the longer the lifetime. Accordingly, toincrease the lifetime of a marker, the depth of a bottom surface of asample well, such as a divot, may extend a certain distance from a metallayer. Also, the materials used to form the sample well can affect thelifetime of the markers. While different markers typically have theirlifetimes shifted in the same direction (e.g., either longer orshorter), the affect may scale differently for different markers.Accordingly, two markers that cannot be distinguished via lifetimemeasurements in free-space may be engineered to be distinguishable byfabricating the sample well environment to adjust the lifetimes of thevarious markers.

A. Lifetime Measurements

Lifetime measurements may be performed using one excitation energywavelength to excite a marker in a sample well. A combination of markershaving distinct lifetimes are selected to distinguish among theindividual markers based on the lifetime measurements. Additionally, thecombination of markers are able to reach an excited state whenilluminated by the excitation source used. An integrated deviceconfigured for lifetime measurements using one excitation may includemultiple pixels positioned along a row where each sample well isconfigured to couple with the same waveguide. A pixel includes a samplewell and a sensor. One or more microcavities or a bullseye grating maybe used to couple the waveguide to the sample well for each pixel.

A pulsed excitation source may be one of the pulsed excitation sourcesusing the techniques described above. In some instances, the pulsedexcitation source may be a semiconductor laser diode configured to emitpulses through direct modulated electrical pumping of the laser diode.The power of the pulses is less than 20 dB of the pulse peak power atapproximately 250 picoseconds after the peak. The time interval for eachexcitation pulse is in the range of 20-200 picoseconds. The timeduration between each excitation pulse is in the range of 1-50nanoseconds. A schematic of how example measurements may be performed isshown in FIG. 31A. Since one excitation energy is used, an excitationfilter suitable for reducing transmission of the excitation energy tothe sensor may be formed in the integrated device, such as thewavelength excitation filter discussed above.

The sensor for each pixel has at least one photosensitive region per apixel. Photons are detected within time intervals of when they reach thesensor. Increasing the number of time bins may improve resolution of therecorded histogram of photons collected over a series of time bins andimprove differentiation among different luminescent markers. When thesensor is configured to detect a particular wavelength, the fourluminescent markers may emit luminescence similar to the particularwavelength. Alternatively, the four luminescent markers may emitluminescence at different wavelengths.

An example set of four luminescent markers that are distinguishablebased on lifetime measurements are ATRho14, Cy®5, AT647N, and CF™633 asshown by the plot in FIG. 31B. These four markers have varying lifetimesand produce distinguishing histograms when at least four time bins areused. FIG. 31C outlines a signal profile for each of these markersacross 16 time bins. The signal profile is normalized for each marker.The time bins vary in time interval in order to provide a unique signalprofile for each of the markers. FIGS. 32A and 32B illustrates signalprofiles, both continuous and discrete, respectively, of anotherexemplary set of markers, ATTO Rho14, D650, ST647, and CF™633, that aredistinguishable based on lifetime measurements. Other sets of markersinclude ATTO Rho14, C647, ST647, CF™633; Alexa Fluor® 647, B630, C640R,CF™633; and ATTO Rho14, ATTO 647N, AlexaFluor647, CF™633.

B. Spectral-Lifetime Measurements

Lifetime measurements may be combined with spectral measurements of oneor more luminescent markers. Spectral measurements may depend on thewavelength of luminescence for individual markers and are captured usingat least two sensor regions per pixel. An exemplary structure of theintegrated device includes pixels that each have a sensor with twodistinct regions, each region configured to detect a differentwavelength. A multi-wavelength filter may be used to selectivelytransmit light of different wavelength to each sensor region. Forexample, one sensor region and filter combination may be configured todetect red light while another sensor region and filter combination maybe configured to detect green light.

Combining both lifetime measurements with spectral measurements may beperformed using one excitation energy wavelength to excite a marker in asample well. A combination of markers is selected having at least twodistinct luminescence wavelengths where the markers emitting at awavelength have distinct lifetimes are selected to distinguish among theindividual markers based on the lifetime and spectral measurements.Additionally, the combination of markers is selected to be able to reachan excited state when illuminated by the excitation source used.

The excitation source is a pulsed excitation source, and may be one ofthe excitation sources using the techniques described above. In someinstances, the pulsed excitation source may be a semiconductor laserdiode configured to emit pulses through direct modulated electricalpumping of the laser diode. The power of the pulses are less than 20 dBthe peak power after 250 picoseconds after the peak. The time durationfor each excitation pulse is in the range of 20-200 picoseconds. Thetime interval between each excitation pulse is in the range of 1-50nanoseconds. A schematic of how example measurements may be performed isshown in FIG. 33A. Since one excitation energy is used, an excitationfilter suitable for reducing transmission of the excitation energy tothe sensor may be used.

The sensor for each pixel has at least two photosensitive regions perpixel. In some embodiments there are two photosensitive regions per apixel. In other embodiments, there are four photosensitive regions per apixel. Each photosensitive region is configured to detect a differentwavelength or range of wavelengths. Photons are detected within timeintervals of when they reach the sensor. Increasing the number of timebins may improve resolution of the recorded histogram of photonscollected over a series of time bins and improve differentiation amongdifferent luminescent markers by their individual lifetimes. In someembodiments, there are two time bins per a region of the sensor. Inother embodiments, there are four time bins per a region of the sensor.

An example set of four luminescent markers that are distinguishablebased on lifetime measurements are ATTO Rho14, AS635, Alexa Fluor® 647,and ATTO 647N. These four markers have two that emit at one similarwavelength and another similar wavelength. Within each pair of markersthat emit at a similar wavelength, the pair of markers have differentlifetimes and produce distinguishing histograms when at least four timebins are used. In this example, ATTO Rho14 and AS635 emit similarluminescence wavelengths and have distinct lifetimes. Alexa Fluor® 647and ATTO 647N emit similar luminescence wavelengths, different from thewavelengths emitted by ATTO Rho 14 and AS635, and have distinctlifetimes. FIG. 33B shows a plot lifetime as a function of emissionwavelength for this set of markers to illustrate how each of thesemarkers is distinguishable based on a combination of lifetime andemission wavelength. FIG. 34A shows a plot of power as a function ofwavelength for ATT Rho14, Alexa Fluor® 647, and ATT) 647N. FIG. 34Bshows plots of fluorescence signal over time for each one of thesemarkers when present in a sample well with a diameter of 135 nm. FIG.35A illustrates the signal profile for these markers across fourphotosensitive regions and each region captures four time bins. Thesignal profiles are normalized and are used to distinguish among thedifferent markers by the relative number of photons captured by aphotosensitive region for each of the four time bins. Other sets of fourfluorophores for such spectral-lifetime measurements are ATRho14, D650,ST647, CF™633; ATTO Rho14, C647, ST647, CF™633; Alexa Fluor® 647, B630,C640R, CF™633; and ATTO Rho 14, ATTO 647N, Alexa Fluor® 647, CF™633.FIG. 35B shows a plot of the signal profile of intensity over time forATRho14, D650, ST647, and C633. FIG. 35C illustrates the signal profilefor ATRho14.

C. Lifetime-Excitation Energy Measurements

Lifetime measurements combined with using at least two excitation energywavelengths may be used to distinguish among multiple markers. Somemarkers may excite when one excitation wavelength is used and notanother. A combination of markers having distinct lifetimes is selectedfor each excitation wavelength to distinguish among the individualmarkers based on the lifetime measurements. In this embodiment, anintegrated device may be configured to have each pixel with a sensorhaving one region and the external excitation source may be configuredto provide two excitation energy wavelengths are electrically modulatedpulsed diode lasers with temporal interleaving.

The excitation source is a combination of at least two excitationenergies. The excitation source is a pulsed excitation source and may beone or more of the excitation sources using the techniques describedabove. In some instances, the pulsed excitation source may be twosemiconductor laser diode configured to emit pulses through directmodulated electrical pumping of the laser diode. The power of the pulsesare 20 dB less than the pulse peak power at 250 picoseconds after thepeak. The time interval for each excitation pulse is in the range of20-200 picoseconds. The time interval between each excitation pulse isin the range of 1-50 nanoseconds. One excitation wavelength is emittedper a pulse and by knowing the excitation wavelength a subset of markerswith distinct lifetimes is uniquely identified. In some embodiments,pulses of excitation alternate among the different wavelengths. Forexample, when two excitation wavelengths are used subsequent pulsesalternate between one wavelength and the other wavelength. A schematicof how example measurements may be performed is shown in FIG. 36A. Anysuitable technique for combining multiple excitation sources andinterleaving pulses having different wavelengths may be used. Examplesof some techniques for delivering pulses of more than one excitationwavelength to a row of sample wells are illustrated are describedherein. In some embodiments, there is a single waveguide per a row ofsample wells and there are two excitation sources that are combined suchthat pulses of excitation energy alternate between the two excitationwavelengths. In some embodiments, there are two waveguides per a row ofsample wells and each waveguide is configured to carry one of twoexcitation wavelengths. In other embodiments, there is a singlewaveguide per a row of pixels and one wavelength couples to one end ofthe waveguide and another wavelength couples to the other end.

The sensor for each pixel has at least one photosensitive region per apixel. The photosensitive region may have dimensions of 5 microns by 5microns. Photons are detected within time intervals of when they reachthe sensor. Increasing the number of time bins may improve resolution ofthe recorded histogram of photons collected over a series of time binsand improve differentiation among different luminescent markers. Thesensor has at least two time bins.

An example set of four luminescent markers that are distinguishablebased on lifetime measurements are Alexa Fluor® 546, Cy®3B, Alexa Fluor®647, and ATTO 647N. As shown in FIG. 36B, Alexa Fluor® 546 and Cy®3Bexcite at one wavelength, such as 532 nm, and have distinct lifetimes.Alexa Fluor® 647 and ATTO 647N excite at another wavelength, 640 nm, andhave distinct lifetimes as shown in FIG. 37A. Distinguishable normalizedsignal profiles across 16 time bins for ATTO647N and CF™633, which areboth excited at 640 nm, are shown in FIG. 37B. By detecting a photonafter a known excitation wavelength, one of these two pairs of markersmay be determined based on the previous excitation wavelength and eachmarker for a pair is identified based on lifetime measurements.

EQUIVALENTS AND SCOPE

Various aspects of the present application may be used alone, incombination, or in a variety of arrangements not specifically discussedin the embodiments described in the foregoing and is therefore notlimited in its application to the details and arrangement of componentsset forth in the foregoing description or illustrated in the drawings.For example, aspects described in one embodiment may be combined in anymanner with aspects described in other embodiments.

Also, the invention may be embodied as a method, of which at least oneexample has been provided. The acts performed as part of the method maybe ordered in any suitable way. Accordingly, embodiments may beconstructed in which acts are performed in an order different thanillustrated, which may include performing some acts simultaneously, eventhough shown as sequential acts in illustrative embodiments.

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

Also, the phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including,” “comprising,” or “having,” “containing,” “involving,” andvariations thereof herein, is meant to encompass the items listedthereafter and equivalents thereof as well as additional items.

1. A method of identifying a single molecule in a sample, the methodcomprising determining a luminescent lifetime and optionally aluminescent intensity of the single molecule.
 2. The method of claim 1,further comprising determining both the luminescent lifetime and theluminescent intensity of the single molecule.
 3. The method of any oneof the preceding claims, further comprising delivering a series ofpulses of one or more excitation energies to the sample.
 4. The methodof any one of the preceding claims, further comprising detecting aplurality of emitted photons from the sample.
 5. The method any one ofthe preceding claims, further comprising recording for each of aplurality of emitted photons from the sample a time duration betweendetection of the emitted photon and a prior delivered excitation energy.6. The method of any one of the preceding claims, wherein identifyingcomprises identifying the single molecule as one type out of a pluralityof possible types of molecules.
 7. The method of claim 6, wherein theplurality of possible types of molecules comprises four types ofmolecules.
 8. The method of claim 7, wherein the four types of moleculescomprise four types of luminescently labeled nucleotides.
 9. The methodof claim 8, wherein the four types of luminescently labeled nucleotidescomprises a first nucleotide comprising adenine, a second nucleotidecomprising guanine, a third nucleotide comprising cytosine, and a fourthnucleotide comprising thymine or uracil.
 10. The method of claim 8,wherein the single molecule is a luminescently labeled nucleotideincorporated into a nucleic acid.
 11. The method of any of the precedingclaims, wherein the method is repeated to identify a series of singlemolecules.
 12. The method of claim 11, wherein the series of singlemolecules is a series of luminescently labeled nucleotides sequentiallyincorporated into a nucleic acid comprising a primer.
 13. The method ofclaim 12, further comprising determining the sequence of a templatenucleic acid by analyzing the sequence of luminescently labelednucleotides incorporated sequentially into the nucleic acid comprisingthe primer.
 14. The method of any one of the preceding claims, whereineach of the plurality of pulses comprises a single excitation energy.15. The method of any one of the preceding claims, wherein a firstportion of the plurality of pulses comprises a first excitation energyand a second portion of the plurality of pulses comprises a secondexcitation energy.
 16. The method of any one of the preceding claims,wherein the series of pulses is delivered at an average frequency ofbetween about 10 MHz and about 100 MHz or between about 100 MHz andabout 1 GHz.
 17. The method of claim 16, wherein the frequency isbetween about 50 MHz and about 200 MHz.
 18. The method of any one of thepreceding claims, wherein for each single molecule identified, thenumber of detected emitted photons is between about 10 and about 100, orbetween about 100 and about 1,000.
 19. The method of any one of thepreceding claims, wherein each single molecule identified is immobilizedon a solid support.
 20. The method of claim 19, wherein the solidsupport comprises a bottom surface of a sample well.
 21. A method ofdetermining the sequence of a template nucleic acid comprising: (i)exposing a complex in a target volume, the complex comprising thetemplate nucleic acid, a primer, and a polymerizing enzyme, to aplurality of types of luminescently labeled nucleotides, wherein eachtype of luminescently labeled nucleotide has a different luminescentlifetime and/or luminescent intensity; (ii) directing a series of pulsesof one or more excitation energies towards a vicinity of the targetvolume; (iii) detecting a plurality of emitted photons fromluminescently labeled nucleotides during sequential incorporation into anucleic acid comprising the primer; and (iv) identifying the sequence ofincorporated nucleotides by determining timing and optionally frequencyof the emitted photons.
 22. The method of claim 21, wherein detectingfurther comprises recording for each detected emitted photon a timeduration between detection of the emitted photon and a prior pulse ofexcitation energy.
 23. The method of claim 21 or 22, wherein each typeof luminescently labeled nucleotide emits photons after beingilluminated by a single excitation energy.
 24. The method of claim 23,wherein the single excitation energy is in a spectral range of about 470to about 510 nm, about 510 to about 550 nm, about 550 to about 580 nm,about 580 nm to about 620 nm, or about 620 nm to about 670 nm.
 25. Themethod of claim 21, wherein one or more type of luminescently labelednucleotide emits photons after being illuminated by a first excitationenergy, and one or more type of luminescently labeled nucleotide emitsphotons after being illuminated by a second excitation energy.
 26. Themethod of claim 25, wherein the first excitation energy is in a spectralrange of about 470 nm to about 510 nm, about 510 to about 550 nm, about550 to about 580 nm, about 580 nm to about 620 nm, or about 620 nm toabout 670 nm, and the second excitation energy is in a spectral range ofabout 470 to about 510 nm, about 510 to about 550 nm, about 550 to about580 nm, about 580 nm to about 620 nm, or about 620 nm to about 670 nm.27. The method of claim 26, wherein the first excitation energy is in aspectral range of about 520 to about 550 nm, and the second excitationenergy is in a spectral range of about 550 to about 580 nm.
 28. Themethod of claim 26, wherein the first excitation energy is in a spectralrange of about 520 to about 550 nm, and the second excitation energy isin a spectral range of about 580 to about 620 nm.
 29. The method ofclaim 26, wherein the first excitation energy is in a spectral range ofabout 520 to about 550 nm, and the second excitation energy is in aspectral range of about 560 to about 670 nm.
 30. The method of claim 26,wherein the first excitation energy is in a spectral range of about 550to about 580 nm, and the second excitation energy is in a spectral rangeof about 580 to about 620 nm. 31-86. (canceled)